Name: measuring physiological responses of drosophila sensory neurons to lipid pheromones using live calcium imaging
Uploaded: 2016-04-29T13:00:00.000+00:00
Duration: 7 min 25 s
Description: Watch this Scientific Journal Video about Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging at JoVE.com

This work was supported by the Singapore National Research Foundation (grant NRF-RF2010-06 to J.Y.Y.).

1.样品制备<ol><li>穿过与GAL4 3,22驱动线UAS-GCaMP5G 19苍蝇（W 1118，P {20XUAS-IVS-GCaMP5G} attP40）。允许跨在25℃生长。 注:生成与Gal4的或UAS-GCaMP转基因的多个拷贝苍蝇可有助于增强荧光信号的强度。当Gr68a-GAL4驱动程序的控制下表达的UAS-GCaMP转基因（UAS-GCaMP3）的早期版本中表现出非常微弱的荧光强度。 <ol><li>收集和性别新eclosed成蝇分开，在25℃的提高。 注:GCaMP5G花期的基线水平在14-28岁之间的日子过得最佳。按性别隔离苍蝇排除可能影响神经反应社会互动的可能性。 </li></ol></li><li>麻醉下的 CO 2的轻微流一只苍蝇。 </li><li>安装在F立法院通过第一把指甲油一小滴在0.17毫米盖玻片中心的玻璃盖玻片。轻轻地放在透明指甲油的滴在其侧面一只苍蝇，并确保整个降由飞覆盖。 </li><li>执行步骤1.5和1.6解剖立体显微镜，在8倍放大下使用。 </li><li>用湿画笔延长苍蝇的前腿。通过将胶带的两个薄条带在第一和第五跗节（ 图2A）固定在位置上的前腿。这将暴露跗节T2-T4用于成像（ 图2B）。 </li><li>要在长鼻图像的神经元，将磁带薄带过长鼻揭露唇瓣（ 图2C，D）的主席台。 </li><li>周围绘制飞疏水屏障用PAP笔，以防止​​在盖玻片表面流动的解决方案。安装的30分钟内进行测量。 </li></ol> 2.刺激的制备解<ol><li>稀释的亲脂性配体（如CH503，本研究中使用）在PBST溶液:137mM的氯化钠，2.7mM的氯化钾，10毫的 Na 2 HPO 4，1.8毫摩尔KH 2 PO 4，0.1％的Triton X-100（V / V ）; pH值7.4。 <ol><li>使用PBST配体的1毫克/毫升储液。准备使用PBST原液的所有后续稀释液。 注意:根据感兴趣的配体的溶解度，可能需要不同的溶剂以溶解配体。 CH503的在各种溶剂中的溶解性在表1中说明。不同的其它溶剂，PBST没有产生荧光的增加。 </li></ol></li></ol> 3.味觉神经元和图像采集的刺激<ol><li>使用10微升移液器，将10微升PBST搬上跗节。 注意:这一步滋润腿部，防止跗节漂流。 </li><li>使用具有苏相机的光学系统fficient速度和灵敏度，实现高时间分辨率良好的荧光信号。 注:在本研究中，我们采用了旋转盘共聚焦显微镜和SCMOS相机（见材料清单 ）。 </li><li>选择特定的跗段和神经元要被成像。收购三家刺激前期共聚焦Z-栈。 <ol><li>理想的情况下，利用采集设置的足够快，以允许最大的时间分辨率，同时还拍摄了整个细胞的体积。例如收购设置为:200毫秒曝光，分档2×2和6×0.5微米2光学切片，捕捉每2秒。 注意:固定化的腿已被成功地成像为至多10分钟，在30秒的时间间隔，用GCaMP信号的没有明显的漂白效果。对于更长的记录时间，确保腿举行非常牢固就位盖玻片上。 </li><li>优化激光功率为最高的信号 - 噪声整个伊马期间以最小的光漂白实现晋政权。对于这里描述的实验中，在30％的透射率使用491纳米二极管激光器（100毫瓦），用于成像的腿和吸管两者Gr68a和ppk23味觉神经元。用2×2像素合并以允许较短的曝光时间，同时仍实现充分的空间分辨率。 注意:激光功率设置进行了优化，使荧光的变化范围从1.2〜4.5倍的检测。 </li></ol></li><li>移取10微升的刺激解到跗节。对于控制实验中，再添10微升的PBST。立即获取117刺激后的图像。 注:获得刺激后的图像数是基于其中在荧光最大变化达到的时间量（约2分钟）。 <ol><li>关键的一步:虽然在准备吸取溶液，不碰飞枪头，因为这将导致前腿搬出位置。 注:另外，用一个显微精确定位含有刺激接近跗段玻璃毛细管将被成像。递送根据制造商的说明使用的细胞内微注射分配器刺激控制卷。 </li></ol></li></ol> 4.图像处理与分析:响应的定量分析<ol><li>打开使用斐济（一系列共聚焦Z-栈<a href="http://fiji.sc/Fiji" target="_blank">http://fiji.sc/Fiji</a> ）23或ImageJ的（ <a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a> ）24图像分析与处理软件。 </li><li>使每个120的时间点的所有六个ž切片的总和强度投影。 <ol><li>在选择"图像"的"堆栈"选项。此外，选择"Z投影"选项，输入"1"作为起始片，"4"为停止片。对于投影类型，选择R20;总和切片"，从下拉菜单中，选择"所有时间框架"。 </li></ol></li><li>通过使用徒手选择工具绘制一个周围细胞体或神经元投射的边界定义感兴趣区域（ROI）的区域。单击"分析"标签，并选择在"工具"下的"投资回报率管理"选项。 </li><li>通过选择"设置测量"选项，"分析"菜单下并检查盒集成密度，面积量化的投资回报率的荧光，平均和最大灰度值和标签。然后，选择投资回报率经理菜单下的"更多"选项，并进一步选择"多措施"选项。 注意:这将产生与每个所选参数的值的弹出框。 </li><li>检查最大灰度值，以确保没有象素已达到饱和（ 例如 ，65,535为一个16位的相机）。 注:有关实验表明在T要么从根据观察到ΔF/ F中的最大增幅，其中细胞体或神经元的预测，衡量他的论文，反应。 </li><li>在时间点t计算荧光（ΔF/ F）的最大相对变化如下: <img alt="式（1）" src="/files/ftp_upload/53392/53392eq1.jpg" /><ol><li>从每个F（单元）值中减去背景荧光。量化背景荧光，从前腿（或吸管）不含可检测的神经元的面积测量等效尺寸和形状的ROI的集成密度。 </li><li>量化的刺激后的荧光，F（细胞）T，测量在刺激后的时间点"t"的细胞体或过程的ROI的集成密度。 </li><li>量化刺激前期荧光，F（细胞）t = 0 时 ，分析细胞体或工艺的刺激前的图像中的方式相同的刺激后信号。使用3个图像的平均积分密度来确定F（细胞）T = 0。 </li></ol></li></ol>

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The authors declare that there are no competing financial interests.

我们在这里描述在2个不同的感觉器官进行果蝇外周神经元的活钙成像的方法。该钙 -evoked由信息素配体CH503呈剂量依赖性和定量诱导Gr68a神经元GCaMP荧光反应。它也可以辨别不同的神经反应模式，如阶段性和补品响应。 显示相位响应神经元被认为可以快速适应不断刺激。这种类型的响应的已用气味探测25和节奏运动活动图形26的生成有关。相比之?...

动物依靠嗅觉和味觉的信息，以调解的生存和繁衍至关重要的决定。理解暗示如何化学感受检测和由神经系统处理要求感官受体（S）和相应的化学配位体的识别。 果蝇检测了惊人的各种挥发性和非挥发性化合物，并且在其中，研究生理极好模型机制的基本chemosensation。而嗅觉器官感知挥发性分子，味觉器官是专门用于检测低挥发性化合物。在这里，我们提出了直接测量果蝇的味觉器官低挥发性，亲脂性配体神经元的反应的方法。 苍蝇的味觉器官包括前腿，喙，和翅膀。分发的味觉器官的表面上是称为感器毛发状结构，其为s响应ugars，苦味，盐，水和费洛蒙8。该感受器已形态分为刷毛的味道和口感钉9。有关于被分类为长（l型）唇瓣约31味刷毛，短（S型）和中间体（i型）的形态。在&#39;L&#39;和&#39;S&#39;是生理糖回应感器房子4感觉神经元（在S细胞），低盐（L1细胞），高盐和苦味化合物（二级电池）和水（在W单元）10 ，11。的"i"感器房屋2的感觉神经元，其中的一个响应既低盐和糖，而其他响应高盐12。有男性约41的味道感受器和分布在每个前肢女26感器。对于男性和女性中，有21感器上midleg，并在后腿13约22感器。由味道感器上腿包围的味觉神经元还包含classified成L1，L2 W和S的类型。 从单个神经元测量电活动的一种标准方法使用外或细胞内的电极来记录离子流。电极测量允许非模式生物进行研究，例如果蝇 ，蛾，和缺乏神经标记广泛的遗传工具蜜蜂神经功能。然而，尽管电生理方法已被经常使用从果蝇味觉测量活动感器4,13,14，运用这种方法存在一些技术挑战。首先，味道刷毛需要基于形态学和空间位置被识别。经鉴定，电测量可通过感受器的尺寸小的阻碍，限制无障碍由于位置，以及难以应用化学刺激的控制卷味道塞得满满的。另外，感器的刺激可以产生一个以上的信号神经型15。其次，电信号检测可以通过从电子设备的机械振动和噪声产生的背景噪音混淆。三，使用锋利的电极会损坏飞行准备，如果使用不当，14。最后，组装电钻机需要刺激递送，信号记录，和数据分析的专门的电子元件，并且可以是昂贵的。 在D.果蝇的基因编码工具的可用性提供了便利的成像技术，使神经元的小种群的反应进行研究开发。一种这样的方法是使用CaLexA记者16。在此方法中，编码的LexA-VP16转录因子和钙敏感蛋白NFAT基因序列融合在一起。的蛋白磷酸酶钙调磷酸酶的钙活化催化NFAT的去磷酸化，并反过来，facilitaTES其导入到细胞核。在细胞核内时，LexA的结构域结合到其引导绿色荧光蛋白（GFP）报道分子的表达，从而使功能性激活的神经元的持续识别LexAop DNA结合基序。该方法已成功地用于测量以下的活苍蝇暴露于臭剂16具体嗅小球中的触角叶的响应。近日，IR52c味觉受体神经元的生理反应，从使用CaLexA 7活苍蝇测量。然而，对于本研究，这是必要的，以年龄苍蝇为长达6周，以提高GFP转录。同时用抗GFP抗体免疫染色可以用于促进CaLexA信号，这种方法将需要组织固定，从而排除了活细胞成像的可能性。 遗传编码的钙指示剂蛋白GCaMP也被广泛用于研究在一些神经元的反应种17。蛋白质GCaMP前神经元的刺激荧光强度低。刺激的应用程序触发的神经元的动作电位，从而导致钙离子的流入。 GCaMP，势必钙 ，经历构象变化，导致它与亮强度（ 图1）发出荧光。最近开发的单神经元钙成像方法被用于鉴定葡萄糖作为配位体的前腿特定Gr61a味觉神经元18。在这项研究中，从转基因果蝇中Gr61a味觉神经元表达GCaMP解剖前肢上覆盖成像之前琼脂糖层。然而，使用解剖组织可引起GCaMP表达的最终破旧，从而限制了测量时间和检测灵敏度。此外，琼脂糖可以限制灵敏度由于荧光的高背景水平及其光散射特性。 ŧO地址一些弊端，我们描述了使用GCaMP介导的钙成像的记录从前肢和长鼻完整的动物的味觉神经细胞的生理反应。我们表明Gr68a的表达遗传编码GCaMP5G 19至果蝇脂质信息素的生理反应和ppk23神经元，（3 的R，11 Z，19 Z）-3-乙酰氧基11,19-octacosadien -1-醇（CH503）20， 21。神经元的反应是通过量化信息素的刺激过程中GCaMP5G信号的荧光增加测量。在这个协议中，神经元被成像为120秒，这足以区分在各个细胞神经元激活模式中的一个总的持续时间。

<table><tbody><tr><td>Gr68a-Gal4</td><td></td><td></td><td>Gift from H. Amrein (Texas A&amp;amp;M Health Science Center, TX, USA) and J. Carlson (Yale University, CT, USA)</td></tr><tr><td>ppk23-Gal4</td><td></td><td></td><td>Gift from K. Scott (Univ. of California, Berkeley, CA, USA)</td></tr><tr><td>UAS-GCaMP5&amp;nbsp;</td><td></td><td>42037</td><td>Bloomington Drosophila&amp;nbsp; Stock Center</td></tr><tr><td>0.17 mm coverslip (Gold-Seal coverslip)</td><td>Electron Microscopy Services</td><td>63790-10</td><td></td></tr><tr><td>Nail polish, &quot;Hard as Nails Clear&quot;&amp;nbsp;</td><td>Sally Hansen</td><td></td><td></td></tr><tr><td>PAP pen</td><td>Sigma-Aldrich&amp;nbsp;</td><td>Z377821</td><td></td></tr><tr><td>Paint brush</td><td></td><td></td><td>fine-tipped brush</td></tr><tr><td>Tape&amp;nbsp;</td><td>Scotch brand</td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich&amp;nbsp;</td><td>13021</td><td></td></tr><tr><td>Ethanol, lab grade</td><td>Merck</td><td>10094</td><td></td></tr><tr><td>Hexane, HPLC grade</td><td>Sigma-Aldrich&amp;nbsp;</td><td>H303SK-4</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich&amp;nbsp;</td><td>472301</td><td></td></tr><tr><td>PBST</td><td></td><td></td><td>Recipe described in the protocol section</td></tr><tr><td>CH&lt;sub&gt;5&lt;/sub&gt;0&lt;sub&gt;3&lt;/sub&gt;</td><td></td><td></td><td>Synthesis described in Mori &lt;em&gt;et al.&lt;/em&gt;, 2010</td></tr><tr><td>sCMOS Camera (ORCA Flash4.0)</td><td>Hamamatsu&amp;nbsp;</td><td>C11578-22U</td><td></td></tr><tr><td>Microscope (Ti-Eclipse)</td><td>Nikon</td><td>Ni-E</td><td></td></tr><tr><td>Spinning Disk Scan head&amp;nbsp;</td><td>Yokogawa</td><td>CSU-X1-A1</td><td></td></tr><tr><td>Aquistion Software (MetaMorph Premier)</td><td>Molecular Devices</td><td>40002</td><td></td></tr><tr><td>Fiji software</td><td>open source</td><td></td><td>&lt;a href=&quot;http://fiji.sc/Fiji&quot;&gt;http://fiji.sc/Fiji&lt;/a&gt;</td></tr></tbody></table>

measuring physiological responses of drosophila sensory neurons to lipid pheromones using live calcium imaging

不同于哺乳动物，昆虫例如果蝇有多个味觉器官。在腿部，长鼻，翅膀和果蝇的产卵的化学感受神经元表达味觉受体1,2，离子通道3-6，和所涉及的检测易失性和非易失性的感官提示的离子型受体7。这些神经元直接联系促味剂，如食品，有毒物质和信息素，因而影响许多复杂的行为如摄食，产卵和配合。电极录音和钙成像已被广泛用于在昆虫量化这些促味剂诱发的神经元的反应。然而，电需要从单一口味感器专用设备和获取测量值可以根据细胞类型，大小和位置在技术上具有挑战性。此外，在果蝇单个神经元分辨率是很困难的，因为味觉感受器后实现本身多于一种类型的化学感受神经元。此处描述的活钙成像法允许测量在活蝇单味觉神经元的响应。这种方法特别适用于成像脂质信息素和其他配体类型的具有在水基溶剂的溶解度低神经元的反应。

使用Gr68a-GAL4或ppk23-GAL4司机GCaMP钙指标进行了基因表达。前脚神经元和非神经细胞支持的不同的种群由每个驱动器（ 图3A-C）标记。在Gr68a-Gal4的观察到亲脂性信息素CH503配体特异性反应和ppk23-Gal4的细胞中表达GCaMP（ 图3D）。在GCaMP5G信号（ΔF/ F）的荧光强度的相对变化，用较高浓度CH503（ 图3E）的增加。有趣的是，CH503的5毫微克?...

Watch this Scientific Journal Video about Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging at JoVE.com

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone.

测量的生理反应<em&gt;果蝇</em&gt;感觉神经元脂质费洛蒙使用实时钙成像

Unlike mammals, insects such as Drosophila have multiple taste organs. The chemosensory neurons on the legs, proboscis, wings and ovipositor of Drosophila ...

measuring-physiological-responses-drosophila-sensory-neurons-to-lipid

Research

JoVE Journal

Immunology and Infection

Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

8.5K 次观看.  Temasek Life Sciences Laboratory. The forelegs and proboscis of Drosophila contain a rich repertoire of gustatory sensory neurons. Here, we present a method using calcium imaging to measure physiological responses from sensory neurons in the foreleg and proboscis of live flies upon exogenous application of a gustatory pheromone. 

视频: Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

载入果蝇与神经末梢钙指标

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

科研

生物学

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory bulb1-5. Olfactory information in the external world is transmitted to the antennal lobe by olfactory sensory neurons (OSNs), which segregate to distinct regions of neuropil called glomeruli according to the specific olfactory receptor they express. Here, OSN axons synapse with both local interneurons (LNs), whose processes can innervate many different glomeruli, and projection neurons (PNs), which convey olfactory information to higher olfactory brain regions.
Optical imaging of the activity of OSNs, LNs and PNs in the antennal lobe - traditionally using synthetic calcium indicators (e.g. calcium green, FURA-2) or voltage-sensitive dyes (e.g. RH414) - has long been an important technique to understand how olfactory stimuli are represented as spatial and temporal patterns of glomerular activity in many species of insects6-10. Development of genetically-encoded neural activity reporters, such as the fluorescent calcium indicators G-CaMP11,12 and Cameleon13, the bioluminescent calcium indicator GFP-aequorin14,15, or a reporter of synaptic transmission, synapto-pHluorin16 has made the olfactory system of the fruitfly, Drosophila melanogaster, particularly accessible to neurophysiological imaging, complementing its comprehensively-described molecular, electrophysiological and neuroanatomical properties2,4,17. These reporters can be selectively expressed via binary transcriptional control systems (e.g. GAL4/UAS18, LexA/LexAop19,20, Q system21) in defined populations of neurons within the olfactory circuitry to dissect with high spatial and temporal resolution how odor-evoked neural activity is represented, modulated and transformed22-24.
Here we describe the preparation and analysis methods to measure odor-evoked responses in the Drosophila antennal lobe using G-CaMP25-27. The animal preparation is minimally invasive and can be adapted to imaging using wide-field fluorescence, confocal and two-photon microscopes.

Calcium Imaging of Odor-evoked Responses in the Drosophila Antennal Lobe

We describe an established technique to measure and analyze odor-evoked calcium responses in the antennal lobe of living Drosophila melanogaster.

在气味诱发反应的钙成像果蝇触角叶

The antennal lobe is the primary olfactory center in the insect brain and represents the anatomical and functional equivalent of the vertebrate olfactory ...

神经科学

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

In Vivo Calcium Imaging of Taste-Induced Neural Responses in Adult Drosophila

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model organism, Drosophila melanogaster. A key strength of this methodology is its ability to record taste-induced neural responses in awake animals without the need for anesthesia. This approach employs binary expression systems (e.g., Gal4-UAS) to express the calcium indicator GCaMP in specific neurons of interest. This protocol describes a procedure in which flies expressing GCaMP are mounted with the labellum securely positioned, enabling fluorescence in the brain to be recorded at millisecond resolution under a confocal microscope while a solution is applied to the labellum, stimulating all labellar taste sensilla. The examples provided focus on calcium responses in primary gustatory receptor neurons of D. melanogaster. However, this approach can be adapted to record from other neurons of interest within the brain of Drosophilids or other insect species. This imaging method enables researchers to simultaneously record collective calcium responses from groups of gustatory neurons across the labellum, complementing electrophysiological tip recordings that quantify action potentials from individual neurons. The in vivo calcium imaging technique outlined here has been instrumental in uncovering molecular and cellular mechanisms of chemosensation, identifying unique temporal response patterns in primary taste neurons, investigating mechanisms of gustatory modulation, and exploring taste processing in downstream circuits.

A calcium imaging approach enables the recording of taste-induced responses in the brains of awake flies while a solution is applied to the labellum. Primary gustatory responses in Drosophila melanogaster are used as an example, but this protocol can be adapted to study downstream neurons or other species.

体内研究成年果蝇味觉诱导神经反应的钙成像

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model ...

In Vivo Calcium Imaging Using Synaptically Localized Calcium Sensors in Drosophila melanogaster

Source: Hancock, C. E. et al., <a href="https://app.jove.com/v/60288">In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster.</a>&#160;J. Vis. Exp. (2019).
This video demonstrates a method for in vivo calcium imaging using synaptically localized calcium sensors in flies. It outlines the steps for preparing the flies for microscopy, conducting olfactory conditioning, and visualizing odor-induced calcium influx.

体内研究在黑腹果蝇中使用突触定位钙传感器进行钙成像

1. Transgenic fruit flies,&#160;Drosophila melanogaster

	Cross female virgin and male flies (raised at 25 &#176;C in 60% relative humidity on a 12 h ...

JoVE Encyclopedia of Experiments

Neuroscience

Neurophysiology

Imaging and Optical Techniques

Drosophila In Vivo Calcium Imaging: A Method for Functional Imaging of Neuronal Activity

Source: Hancock, C. E., et al. <a href="https://www.jove.com/video/60288/" target="_blank">In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster</a>. J. Vis. Exp. (2019).
This video describes in vivo calcium imaging in Drosophila neurons using GCaMP. The featured protocol clip shows how to record changes in GCaMP fluorescence from mushroom body neurons during an olfactory conditioning experiment.

体内果蝇钙成像:一种神经元活动功能成像的方法

Source: Hancock, C. E., et al. In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster. J. Vis. Exp. (2019).
...

黑腹果蝇（果蝇）

成虫

Detecting Physiological Responses from Sensory Neurons in Drosophila

Source: Shankar, S., et al. <a href="https://app.jove.com/v/53392">Measuring Physiological Responses of Drosophila Sensory Neurons to Lipid Pheromones Using Live Calcium Imaging.</a> J. Vis. Exp. (2016).
This video demonstrates a method for detecting sensory neuron responses in Drosophila using ligand binding and fluorescence imaging. Ligands bind to neuron receptors, causing calcium influx. The calcium binds to indicator complexes, leading to fluorescence emission, which confirms the neuronal response to the ligand.

检测果蝇感觉神经元的生理反应

1. Sample Preparation

	Cross the Gal4 driver line to UAS-GCaMP5G Drosophila melanogaster flies (w;P{20XUAS-IVS-GCaMP5G}attP40). Allow the cross to grow ...

测量的生理反应

摘要

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