我们感谢T.Stürner对稿件有益的修正，讨论和评论; SL Zipursky提供即时的股票;中号Schölling用于进行图像处理。图像分析部分A处Kakita的实验室中进行。这项工作是由亚历山大·冯·洪堡基金会和日本学术振兴会奖学金为研究海外（AS），日本学术振兴会研究员（SH-S），格兰特 - 在 - 援助启动（24800024），对创新领域（25110713），持田的支持，武田，稻盛和夫，第一三共，东丽基金会（TS），DZNE核心资金（GT）和DZNE光学显微镜基金（CM）。

在这个协议中所述的实验程序涉及专门与果蝇工作，并不会受到在德国和日本动物福利法。 1.光照条件<ol><li>准备携带sensless-翻转 （SENS-FLP）和bruchpilot-FRT-STOP-FRT-GFP（BRP-FSF-GFP）15在感光R8神经元（R8S）可视化BRP-GFP苍蝇。一个细菌人工染色体（BAC）中的基因组基因座编码BRP被修改以产生此BRP-FSF-GFP构建体。在执行该修改的BAC BRP-GFP表达的蝇是内源调节序列的控制之下，但sensless-翻转酶介导的切除FRT-STOP-FRT盒15的后限于R8光感受器。需要注意的是SENS-FLP很少表达在R7S翻转酶。 </li><li>不让苍蝇在12小时里GHT / 12小时黑暗周期（LD），在25℃下从幼虫阶段，直至羽化（ 图1A）。 </li><li>收集苍蝇0 - 6小时羽化后，将它们放入含有飞食品新的小瓶。 </li><li>设置小瓶与三个步骤（41厘米的高度，21厘米的的基础上，其厚度为4厘米，13厘米的每个步骤的高度）（ 图1B）由丙烯酸树脂构成的透明的机架。 </li><li>调整齿条和一个LED面板之间的距离，以提供照明用的数字光计（ 图1C）1000勒克斯的平均强度。 </li><li>保持1苍蝇- 3天以下条件之一:持续黑暗（DD），12小时光照/ 12小时黑暗周期（LD），或恒光（LL）在25℃的小型孵化（ 图1A和1C）。 </li></ol> 2.解剖，免疫组化和成像<ol><li>解剖大脑飞用钳子，用4％FO修复rmaldehyde和免疫染色用抗Chaoptin第一抗体（1:50）和用于根据以前的实验程序25感光体的可视化的第二抗体。简述: <ol><li>制备0.1％的Triton X-100的PBS（0.1％PBT）。该溶液是用来改善抗体的组织穿透。 </li><li>麻醉苍蝇在二氧化碳垫，整理出正确的基因，并将它们转移到一个培养皿中充满0.1％PBT。 </li><li>粘钳子长鼻下方并分别与其他钳拉断右眼和左眼。 </li><li>除去长鼻和连接到大脑气管。 </li><li>与镊子进入含有0.1％的PBT 150微升在RT 1.5mL管转移大脑。 </li><li>距离2.1.3 10分钟重复 - 2.1.5，以获得尽可能多的大脑越好。 </li><li>添加16％的甲醛50μL进管吹打混合。 </li><li>孵育大脑在室温固定液温度50分钟。 </li><li>用0.1％的PBT 200微升洗涤三次用微量，注意离开的大脑中的管中。 </li><li>在最后一次洗涤后，取出0.1％PBT和添加第一抗体溶液，制备如下:添加0.3％的PBT（0.3％的Triton X-100的PBS）抗Chaoptin抗体的4微升（最终稀释1的196微升:50），用于光感受器的可视化。 </li><li>孵育在一次抗体溶液的大脑在4℃CO / N。 </li><li> 3倍洗净用微量200微升0.1％的PBT。 </li><li>最后一次洗涤后，除去0.1％的PBT，并添加含二级抗体的0.3％PBT的200微升。 </li><li>在4°CO / N孵育它在黑暗中。 </li><li>与用微量200μL的0.1％的PBT洗涤三次。 </li></ol></li><li>安装时，把两个小滴的安装介质（每次2μL）用微量在显微镜载玻片中心在约两厘米见方DISTANCE彼此。 </li><li>将两个盖玻片，一是到每个下降，留下的约为0.2毫米盖玻片之间的狭窄缝隙。 </li><li>放置在盖玻片顶部的安装平台15微升并用微量添加的大脑到安装平台。 </li><li>在解剖显微镜下，用腹侧定位大脑成（ 图2）在两个盖玻片之间的窄的间隙。 </li><li>在上面放置盖玻片并密封用透明指甲油固定检体（ 图2）的盖玻片的边缘。 </li><li>获得脑部图像用共聚焦显微镜。使用63X油浸物镜（1.4 NA）和2.6倍数码变焦。产生第二视神经节髓质超过20光学切片为0.5微米的步长大小。 </li></ol> 3.斑点，表面对象，开始点和结束点的生成<ol><li>量化数，颇ibution和BRP-GFP泪点的离域电平，打开在图像分析软件通过共聚焦显微镜获得的协议栈和重建的3D图像（ 图3A）。注意，在此研究中的步骤，用了Imaris进行。 </li><li>通过为每个R8轴突终端斑检测模块识别BRP-GFP泪点。 <ol><li>选择"添加新景点"。 </li><li>在算法设置中选择"段只是一个感兴趣区域"。选择手动髓质的R8轴突终末。 </li><li>选择BRP-GFP信号的源信道。 注:R7和R8感光体轴突与抗Chaoptin（ 图3A）免疫标记。仅R 8，不过，包含BRP-GFP泪点并因此可以很容易地区别（ 图3A）。另外，R8的端点可以通过抗Chaoptin阳性轴突在入口点到M3的髓质层变薄来识别。 </li><li>设置估计XY面直径TØ0.35微米，并在现场检测勾选"后台减法"。 </li><li>选择滤波器类型"质量"，并自动过滤。然后单击Finish。 </li><li>从3.2.1 T-3.2.5重复其它R轴突（ 图3B）。 </li></ol></li><li>为了测量BRP-GFP的R8轴突离域层面，以及每个轴突（ 图3C）的"表面"功能生成曲面对象。 <ol><li>选择"添加新的表面"。 </li><li>在算法设置中选择"段只是一个感兴趣区域"。然后手动选择在髓质的R8轴突终末。 </li><li>选择感光器的源通道。 </li><li>勾选"平滑"。 </li><li>在门槛，选择"绝对强度"。 </li><li>勾选"启用"。 "种子点直径"为0.5微米。 </li><li>选择滤波器类型"质量"和"素数"，然后筛选automati凯莉。 </li><li>进入"编辑"，并删除其他非有趣的轴突产生表面的碎片。 </li><li>从3.3.1重复- 3.3.8其它R轴突（ 图3C）。注意一些表面的物体被打断，因为相对较小，且在M2层的光感受器的微弱信号，但他们仍然认为作为一个对象从开始到结束点。 </li></ol></li><li>以确定BRP-GFP泪点的沿着在髓质神经毡每个神经元的分布，定义起始点和结束点与所述测量点的功能（ 图3D）。 <ol><li>选择"添加新的测量点"。然后选择"编辑"，然后选择"对象表面"与表面物体的顶部相交。 </li><li>把测量点上的M1层R.轴突的表面物体的顶部顺序。这些现在被定义为开始点（ 图3D）。 </li><li>选择"添加新的测量点"。然后选择"编辑"，然后选择"对象表面"与表面物体的底部相交。 </li><li>把测量点上在M3层R.轴突的表面的物体的底部的顺序。这些现在被定义为结束点（ 图3D）。 </li></ol></li><li>减去来自GFP的信道的背景信号，产生表面的对象，地点，开始点和结束点为M6的层（ 图3E）两个R轴突。 <ol><li>选择"添加新的表面"。 </li><li>在算法设置中选择"段只是一个感兴趣区域"。然后手动选择M5和M6层之间的R7轴突终末。 </li><li>选择感光体的源通道。 </li><li>勾选"平滑"。 </li><li>在门槛，选择"绝对强度"。 </li><li>选择滤波器类型"质量"和"素数"，然后自动过滤。 </li><li>选择"添加新景点";。 </li><li>选择"跳过自动创建，手动编辑"。 </li><li>选择"对象的中心"，并在从3.5.1中产生的表面的对象的内部区域加一个点 - 3.5.6。 </li><li>从3.5.1重复 - 3.5.9另一个R7轴突终末。 </li><li>选择"添加新的测量点"。然后选择"编辑"，并把在M6的层中的R 7轴突终端的尖端的两个表面的物体的顶部测量点。现在，这些均定义为启动点。 </li><li>选择"添加新的测量点"。然后选择"编辑"，并把在M6的层中的R 7轴突终端的尖端的两个表面的物体的底部测量点。这些现在被定义为结束点（ 图3E）。 </li></ol></li></ol> 4.计算点的数量的，现货的频率和分布BRP-GFP的富集程度与一个自定义图像数据分析软件<ol><li>复制文件"XtquantifySynapseSNR.m"和"功能"文件夹进了Imaris XT MATLAB的文件夹中。注意，在此研究的计算用在Matlab实现的定制的插件执行。从网站（下载插件<a href="https://github.com/cmohl2013/synapse_trafo" target="_blank">https://github.com/cmohl2013/synapse_trafo</a> ）。 </li><li>打开一个数据集有n个点，正表面和2个测量点的对象，每个都包含n个测量点（3.1编写 - 3.4）。第一个测量点对象定义为所有轴突的起始位置。第二个测量点的对象定义了所有轴突的结束位置。两个测量点对象需要具有相同数量的测量点（即轴突的数量）。 </li><li>执行图像处理&gt;&gt; CUSTOM&gt; atsushis突触检测VS 6。 </li><li>检查元数据和更改像素大小的图像分析软件的情况下，这是不正确的。 </li><li>对于PERF选择通道斑点区域和胞浆区域（与BRP-GFP的信道）的强度分析ormance。 </li><li>定义文件名导出的结果。 </li><li>定义百分比仓和轴突长度的数目。输入仓为"10"和箱为"100％"（ 图4A和4B）的范围的数量。 </li><li>定义点和其它细胞质区域的BRP-GFP离域分析的区域。输入点半径为"0.35微米"和周边地区为"50微米"的宽度。斑点半径定义了泪点GFP信号（直径0.35微米，集中在一个泪点）的区域。周边区域的宽度定义区域在细胞质区域的GFP信号（直径50微米，集中于泪点，但仅包括R 8和不含在细胞质中的所有点区域）（ 图4A和4C）。 </li><li>等到所有的神经元的口罩被处理并导出为PDF文件（ 图4B和4C），并在电子表格中进一步分析CSV表。该CSV表包括斑点数，在"胞质区域"，并且在每个块对所有R8轴突的"斑点"的最大信号强度的平均信号强度。 </li><li>打开3.5编写的背景信号的减法的数据集。 </li><li> 4.9 - 从4.3的描述执行图像处理。 </li><li>计算的总数的平均值和BRP-GFP泪点的电子表格中的（ 图4D和4E）的分布。 </li><li>由平均强度中的"胞质区域"通过的最大强度在"点"的平均强度在背景信号中减去分割比率计算BRP-GFP信号的离域的电子表格中的平均值（ 图4F） 。 </li></ol>

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<li>Mosca, T. J., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptic+organization+of+the+Drosophila+antennal+lobe+and+its+regulation+by+the+Teneurins%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins.</a> Elife. 3, e03726(2014).</li>
<li>Wu, J. S., Luo, L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/17487202">A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining.</a> Nat Protoc. 1 (4), 2110-2115 (2006).</li>
<li>Fischbach, K. F., Dittrich, A. P. M. <a target="_blank" href="http://link.springer.com/article/10.1007/BF00218858">The Optic Lobe of Drosophila-Melanogaster .1 A Golgi Analysis of Wild-Type Structure.</a> Cell and Tissue Research. 258 (3), 441-475 (1989).</li>
<li>Berger-Muller, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessing+the+role+of+cell-surface+molecules+in+central+synaptogenesis+in+the+Drosophila+visual+system%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Assessing the role of cell-surface molecules in central synaptogenesis in the Drosophila visual system.</a> PLoS One. 8 (12), e83732(2013).</li>
<li>Fouquet, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maturation+of+active+zone+assembly+by+Drosophila+Bruchpilot%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Maturation of active zone assembly by Drosophila Bruchpilot.</a> J Cell Biol. 186 (1), 129-145 (2009).</li>
<li>Ke, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Super-Resolution+Mapping+of+Neuronal+Circuitry+With+an+Index-Optimized+Clearing+Agent%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent.</a> Cell Rep. 14 (11), 2718-2732 (2016).</li>
<li>Ehmann, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+super-resolution+imaging+of+Bruchpilot+distinguishes+active+zone+states%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states.</a> Nat Commun. 5, 4650(2014).</li>
</ol>

The authors have nothing to disclose.

在这项研究中，我们展示了如何准备光照条件下暴露苍蝇平等的光强度。那么，定量突触标记泪点的数量，但也可以在空间上解决突触沿轴突密度，并测量在细胞质区域的标志物蛋白的离域电平。这三个评估允许我们在不同环境条件下的单神经元水平评价突触动力学的细节。我们的协议可以适应不同的突触蛋白，而且对包含点状的信号的任何数据。 该协议中的关键步骤是大?...

<html>突触功能的调制有助于神经系统的显着能力精确响应或适应不断变化的环境刺激。调节突触前小泡释放的概率是控制突触强度1的一种方法。突触囊泡释放在活动区（AZ），突触前膜2的一个专门区域发生。在AZ的特征在于特定蛋白质3 4的盒，。促进AZ组装大多数蛋白质是高度保守的线虫，昆虫和哺乳动物5。最近的研究表明，神经元活动的水平调节AZ，这又在体外和体内 6,7有助于官能输出的调节的分子组成，&gt; 8。我们以前发现，感光AZS发生在果蝇分子重塑长时间暴露在自然的环境光9点以后。在这种情况下，我们观察到Bruchpilot（BRP）阳性AZS的数量在感光体轴突降低。 该BRP / CAST / ELKS家族蛋白是脊椎动物和无脊椎动物神经突触10 AZS的基本构件。在果蝇突变BRP，诱发囊泡释放被抑制11，12。 BRP的17 C-末端氨基酸残基是在果蝇神经肌肉接头（NMJ）13,14突触小泡簇的关键。这些研究证明这种分子在亚利桑那州的组织和功能的核心作用。与最近开发的基因工具，突触标记与重组（STAR），BRP可以在特定细胞类型的体内观察到的，在内源表达水平和在单个突触决议15。这个工具使得它可行的定量评价在复杂的中枢神经系统突触的内生动力。 已经有多项研究，包括基于从共聚焦显微镜获得的数据突触quantifications。突触改动了测量的长度，面积，体积，密度和基于复杂的软件应用计数的数目进行评价。例如，免费软件ImageJ的提供了用于总突触面积，并在果蝇 NMJ 16突触密度措施定量方法。前期和突触后标记的共定位网站的数量一直在使用插件"泪点分析仪的"ImageJ的软件平台17可量化。替代地，多参数adigm数值计算环境的基础方案，突触检测器（SynD），可以自动跟踪标记有荧光标记的神经元的树突，然后量化突触蛋白水平作为距离从细胞体18的功能。软件突触泪点分析（SynPAnal），已被设计用于从共焦或荧光显微镜获取的神经元的二维图像的快速分析。该软件的主要功能是蛋白质泪点19的密度和强度的自动和快速定量。近日，在3D突触20多项量化已生成一个自动基于学习的突触检测算法，采取的三维可视化辅助分析（Vaa3D）软件21的优势。 商业图像分析软件也是突触quantifications的强大工具。例如，荧光标记的神经递质受体或突触前AZ组件已在三个维度与C中的单突触分辨率线虫 22或果蝇嗅觉系统23，24，使数百突触被快速地特征在于单个样品中进行定量。 这里，我们通过定制图像分析软件呈现一个方法插件在一个多范式数值计算环境，允许分析AZS的半自动多个方面，包括它们的数量，分布和分子成分浓缩到水平实现在AZ。因此，这种复杂的分析使我们能够评估在不同环境条件下轴突终末突触部件的动力学。我们研究了成年果蝇光感受器的输出突触曝光的效果。这个程序是在三个步骤执行:1）对于光照射，2）夹层，免疫组织化学和共焦成像，3）图像分析制剂。

<table><tbody><tr><td>Vial</td><td>Hightech, Japan</td><td>MKC-20</td><td></td></tr><tr><td>Plug</td><td>Thermo Fisher Sciehtific, USA</td><td>AS-275</td><td></td></tr><tr><td>Customized transparent rack made of acrylic resin&amp;nbsp;</td><td>Shin-Shin Corporation, Japan</td><td></td><td>a height of 41 cm, a base of 21 cm, a thickness of 4 cm and a height of 13 cm for each step</td></tr><tr><td>Cool incubator</td><td>MITSUBISHI ELECTRIC, Japan</td><td>CN-40A</td><td></td></tr><tr><td>LED panel</td><td>MISUMI, Japan</td><td>LEDXC170-W</td><td></td></tr><tr><td>Digital light meter</td><td>CEM</td><td>DT-1301</td><td></td></tr><tr><td>Fly pad</td><td>Tokken, Japan</td><td>TK-HA03-S</td><td></td></tr><tr><td>Petri dish (35 x 10 mm)</td><td>Greiner Bio-One International, Germany</td><td>627102</td><td></td></tr><tr><td>PBS tablet</td><td>Takara, Japan</td><td>T900</td><td></td></tr><tr><td>Triton X-100</td><td>Wako, Japan</td><td>160-24751</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>1.5 mL tube</td><td>Sarstedt, Germany</td><td>A. 152X</td><td></td></tr><tr><td>Formaldehyde 16%</td><td>NEM, Japan</td><td>3152</td><td></td></tr><tr><td>Pipetman P-200</td><td>Gilson</td><td>F123601&amp;nbsp;</td><td></td></tr><tr><td>Pipetman P-20</td><td>Gilson</td><td>F123600</td><td></td></tr><tr><td>Pipetman P-2</td><td>Gilson</td><td>F144801</td><td></td></tr><tr><td>anti-chaoptin antibody</td><td>DSHB</td><td>24B10</td><td></td></tr><tr><td>Alexa568-conjugated anti-mouse antibody</td><td>Life Technologies</td><td>A-11031</td><td></td></tr><tr><td>VECTASHIELD Mounting Medium</td><td>Vector Laboratories, Inc.</td><td>H-1000</td><td></td></tr><tr><td>Microscope slide (76 x 26 mm)</td><td>Thermo Fisher Scientific Gerhard Menzel B.V. &amp;amp; Co. KG, Germany</td><td></td><td></td></tr><tr><td>Coverslip (18 x 18 mm, 0.17 mm)</td><td>Zeiss, Germany</td><td>474030-9000-000</td><td></td></tr><tr><td>Industrial Microscopes</td><td>Olympus, Japan</td><td>SZ61-C-SET</td><td></td></tr><tr><td>Stereo Microscope Lighting</td><td>Olympus, Japan</td><td>KL 1600 LED</td><td></td></tr><tr><td>confocal microscopy</td><td>Zeiss, Germany</td><td>LSM780</td><td></td></tr><tr><td>Imaris</td><td>Bitplane, Switzerland</td><td></td><td>Version 7.6.4 or above</td></tr><tr><td>Matlab</td><td>The MathWorks, Inc., USA</td><td></td><td></td></tr><tr><td>Excel for Mac&amp;nbsp;</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

analyzing synaptic modulation of drosophila melanogaster photoreceptors after exposure to prolonged light

神经系统有适应和各种刺激作出反应的非凡能力。这种神经调节主要是在突触水平通过可塑性来实现的。活动区（亚利桑那州）是在该介导神经递质的释放，并且由支架蛋白的致密集合的突触前膜的区域。 果蝇的锆刚玉（ 果蝇 ）光感受器长时间暴露在自然光环境发生后重塑分子。因而神经元活性的水平可以重新排列AZ的分子组成和向功能输出的调节。 从曝光设置准备到免疫组开始，该协议细节如何量化数，空间分布，并且在AZS 果蝇光感受器突触分子的离域电平。使用图像分析SOFtware，绿色荧光蛋白融合AZ成分Bruchpilot集群被认定为每个R8感光器（R8）轴突终端。检测Bruchpilot点被自动分配给各个R8轴突。要计算点频率沿轴突的分布，我们实现了一个定制的软件插件。每个轴突的开始点和结束点进行人工定义和每个Bruchpilot光斑的位置被投影到的开始和结束点之间的连接线。除了Bruchpilot簇的数目，我们还定量Bruchpilot-GFP的簇内的离域电平。这些测量反映详细在单个神经元的空间分辨突触动力学不同环境条件下对刺激。

果蝇的复眼包括〜780小眼，每个包含八种类型光感受器（R1-8）。 R7和R8项目它们的轴突到第二视神经节，髓质，在那里它们形成在层M6和M3，分别为26突触。为了研究长时间暴露于光的感光R8活动区域的分子组成的影响，我们采取了STAR方法15的优势。 BRP的内源性表达水平荧光翻转出BRP-FSF-GFP终止密码子在从SENS-FLP衍生R8...

Watch this Scientific Journal Video about Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light at JoVE.com

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

在这里，我们将展示如何量化的数量和果蝇光感受器突触活动区域的空间布局，突出了遗传编码的分子标记，并长时间暴露于光后的调制。

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

analyzing-synaptic-modulation-drosophila-melanogaster-photoreceptors

Research

JoVE Journal

Neuroscience

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

6.5K 次观看.  Niigata University. 在这里，我们将展示如何量化的数量和果蝇光感受器突触活动区域的空间布局，突出了遗传编码的分子标记，并长时间暴露于光后的调制。 

视频: Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

科研

生物学

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

光偏好的检测，以研究的先天和昼夜调节感光特性<em&gt;果蝇</em&gt;幼虫

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

神经科学

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

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, ...

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 ...

Behavioral Assays for Optogenetic Manipulation of Neural Circuits in Drosophila melanogaster

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study introduces easy-to-implement setups for applying optogenetic methods in Drosophila melanogaster. Two optogenetic tools, CsChrimson, a red-light-activated cation channel, and GtACR2, a blue-light-activated anion channel, were employed in four experimental approaches. Three of these approaches involve single-fly experiments: (1) a blue-light optogenetic thermotactic positional preference assay targeting temperature-sensitive heating cells, (2) a red-light optogenetic positional preference assay activating bitter sensing neurons, and (3) a proboscis extension response assay activating the sweet-sensing neurons. The fourth approach (4) is a fly maze setup to assess avoidance behaviors using multiple flies. The ability to manipulate neural activity temporally and spatially offers powerful insights into sensory processing and decision-making, underscoring the potential of optogenetics to advance our knowledge of neural function. These methods provide an accessible and robust framework for future research in neuroscience to enhance the understanding of specific neural pathways and their behavioral outcomes.

This paper presents methods for optogenetic manipulation in Drosophila melanogaster, utilizing CsChrimson and GtACR2 to activate and silence specific neurons. Four experiments are described to utilize optogenetics to explore thermotactic and gustatory behaviors, providing insights into the underlying neural mechanisms governing these processes.

黑腹果蝇神经回路光遗传学作的行为测定

Optogenetics has become a fundamental technique in neuroscience, enabling precise control of neuronal activity through light stimulation. This study ...

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 ...

JoVE Encyclopedia of Experiments

Neurophysiology

Stimulation and Modulation Techniques

Synaptic Modulation in Drosophila After Exposure to Prolonged Light

Source: Sugie, A., et al. <a href="https://app.jove.com/v/55176">Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light.</a> J. Vis. Exp. (120)
This video demonstrates the rearing of Drosophila melanogaster under specific light conditions to study the role of Bruchpilot proteins in neurotransmitter release and the resulting modulation of synaptic activity.

果蝇长时间暴露于光照后的突触调节

1. Light Exposure Conditions

	From the larva stage until eclosion, keep the flies in a 12 h light/12 h dark cycle (LD) at 25 &#176;C (Figure 1A).
	...

Analyzing Synaptic Vesicle Recycling and Fluorescent Dye Uptake in Drosophila Larvae

Source: Kopke, D. L., et al., <a href="https://app.jove.com/v/57765">FM Dye Cycling at the Synapse: Comparing High Potassium Depolarization, Electrical and Channelrhodopsin Stimulation.</a> J. Vis. Exp. (2018).
This video demonstrates a protocol to study synaptic vesicle recycling at neuromuscular junctions in channelrhodopsin-expressing Drosophila larvae using a fluorescent dye. Blue light activates the channelrhodopsins, causing calcium influx and synaptic vesicle exocytosis. The dye integrates into the neuronal membranes and is internalized during endocytosis, resulting in enhanced fluorescence. Further blue light stimulation induces exocytosis and dye release, which is shown by reduced fluorescence.

分析果蝇幼虫的突触囊泡循环和荧光染料摄取

1. Larval Glue Dissection

	Thoroughly mix 10 parts of silicone elastomer base with 1 part of silicone elastomer curing agent from the elastomer kit ...

分析突触调制

摘要

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Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

光偏好的检测，以研究的先天和昼夜调节感光特性

发展和成人的制备果蝇大脑和视网膜实时成像

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

测量果蝇光感受器中光色素水平的电生理学方法

黑腹果蝇神经回路光遗传学作的行为测定

研究果蝇光感受器中的延长去极化后电位（PDA）

果蝇长时间暴露于光照后的突触调节

分析果蝇幼虫的突触囊泡循环和荧光染料摄取

分析突触调制

摘要

探索更多视频

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

光偏好的检测，以研究的先天和昼夜调节感光特性

发展和成人的制备果蝇大脑和视网膜实时成像

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

测量 果蝇 光感受器中光色素水平的电生理学方法

黑腹果蝇神经回路光遗传学作的行为测定

研究果蝇光感受器中的延长去极化后电位 （PDA）

果蝇长时间暴露于光照后的突触调节

分析果蝇幼虫的突触囊泡循环和荧光染料摄取

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）