Name: analyzing synaptic modulation of drosophila melanogaster photoreceptors after exposure to prolonged light
Uploaded: 2017-02-10T14:00:00
Description: Watch this Scientific Journal Video about Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light at JoVE.com

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?...

<ol>
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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#160;</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. &#38; 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>
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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 ...

The overall goal of this experimental procedure is to understand synaptic dynamics in a single neuron under different activation conditions. This method can help answer key questions in the synaptic plasticity field such as revealing changes in the molecular composition of synapses upon maturation of the neuron's activity. The main advantage of this technique is that it allows the semi-automated analysis of multiple aspects of synapses including their number, distribution, and the level of enrichment of molecular components after synapses.For this experiment, collect the flies into normal vials within six hours of eclosion. Load the collection vials into a transparent acrylic rack. In a small incubator set to 25 degrees celsius, position the rack at a precise distance from an LED panel where the light exposure is at an average of 1000 lux.Then, rear flies for one to three days using one of the following conditions, either constant darkness, 12 hours of light followed by 12 hours of darkness, or constant light. Later, dissect and stain the brains using standard techniques. To mount the fly brains, load a micropipet with mounting medium and deposit two 2 microliter drops at the center of a microscope slide about 2 cm apart.Place a cover slip onto each drop and the gape between about 0.2 mm. Then deposit 15 microliters of mounting medium over the cover slips and into the gap. Under a dissecting microscope, deposit the brain into the gap from micropipet.And then position the brains, ventral side up. Finally, attach a cover slip and seal it along the edges using clear nail polish. The brains can then be imaged using standard techniques to reconstitute 3D images.In this case, GFP luminescence is documented in cells with active Brp expression using the star method. Also in this example, R7 and R8 photoreceptor axons were immunolabled with anticiaoptin, which was viewed using RFP-tagged secondary antibodies. To quantify the number, distribution, and delocalization level of the Brp GFP puncta first load reconstituted 3D images of the brain made from image stacks.Brp puncta are displayed in white. Now, find the region of interest. In this case, R8 axon's terminals are located by following the thinning of the anticiaoptin positive axons at the entry point into the M3 medulla layer.At each R8 axon terminal identify the Brp GFP puncta using the spot detection module. Select add new spots then select segment only region of interest in the algorithm settings. Once the analysis region is defined, set the source channel to Brp GFP then set the estimated XY diameter to 0.35 microns.And then, check off background subtraction in the spot detection. Next, select quality as the filter type, and the puncta are filtered automatically. Complete this step by clicking finish.Repeat the spot detection method for all of R8 axons in the dataset. The next region to identify is the axon cytoplasm. First, generate surface objects with the surface function.Then, select add new surfaces, and under the algorithm settings, toggle segment only region of interest. Now, manually select the region of interest. Next, set the calculation perimeters.To find the photoreceptor channel as source, select the smooth option. For the threshold, select absolute intensity. Enable the split touching objects option.Set the seat points diameter to 0.5 microns and use the default filter settings for quality and number of voxels. The filter is then applied automatically. Now, go to edit and delete the pieces of surface generated outside of the region of interest.In this case, the other axons. After repeating the axon region detection for all R8 axons in the dataset, all the Brp puncta and R8 axons are identified as a set of spot objects. And the corresponding cytoplasmic regions are identified as surface objects.Now, proceed by manually defining each axon's orientation and space. This is important for later quantification of Brp GFP density along each neuron in the medulla neuropil. To do this, first define their start points and end points.Select add new measurement points, then select edit followed by select surface of object to work with the top of the surface objects. To define the start points, put measurement points on all the surface objects of the R axons in the M1 layer. Place these points in a systematic reproducible order.Next, define the end points. Select add new measurement points, edit, and surface of object again. Then, repeating the same systematic order, place measurement points on the bottom of the R axons in M3 layer.To quantify the Brp background intensity, analyze the cytoplasmic regions of two R7 axons. Select add new surfaces and manually define the region of an R7 axon as was done for the R8 axons. Use identical settings except do not toggle on the enable split touching objects option.Leave this off. Next, add a dummy spots object inside a defined R7 axon region. Select add new spots, and select skip automatic creation, and edit manually.Then, select center of object and click on the surface object to place the dummy spots object inside the R7 axon. Now, repeat the surface and spot detection steps for a second R7 axon. And then define the start and end terminal points of the R7 axons as done with the R8 axons.For this analysis, be certain to have the correct software installed. Open the dataset containing the spot data, surfaces data, and two measurement point objects, each containing the same number of measurement points. Inspect the data.Both of the measurement point objects must have the same number of measurement points for the calculations to work. Once the spots, axon regions, and start and end points have been defined, start the plugin. First, check the metadata, in particular, the voxel size.Open image properties from edit, then check the 3 dimensions of the voxel and microns under coordinates in geometry. Next, select a channel for the intensity analysis. In this case, the channel showing Brp GFP is selected.Then define the file name for the results. Next, define the number of bins as 10, and set the axon length to 100%Then define the regions of the puncta by setting the spot radius to 0.35 microns and the surrounding cytoplasmic region to 50 microns. Finally, execute the synapse detection command, and later, statistically analyze the output.Following the described protocols, Brp GFP puncta were analyzed in the R8 synapses of flies exposed to constant darkness, constant light, or a normal light/dark cycle. The number of Brp puncta was significantly reduced in R8 photoreceptors of flies kept in constant light. The distribution of the puncta was calculated using a custom plugin.R8 synapses were distributed all along the axonal shaft from the M1 to the M3 layer, but their density was higher at the M1 and M3 layers. Similarly, delocalization levels of the puncta were calculated. They were unchanged in all conditions, however delocalization levels of Brp GFP differed from other reporters, like Brp-short-cherry which becomes clearly defused under constant light.It is possible that there is inappropriate processing of the Brp short fragment after disassembling from the AZ.After watching this video, you should have a good understanding of how to analyze synaptic plastic properties in a single neuron. Such plastic properties includes the synapse number, their distribution, and the label of enrichment of a specific molecular component after the synapses.

Research

JoVE Journal

Neuroscience

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

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

科研

神经科学

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

Determination of the Spontaneous Locomotor Activity in Drosophila melanogaster

在自发活动的测定<em&gt;果蝇</em

Drosophila melanogaster has been used as an excellent model organism to study environmental and genetic manipulations that affect behavior. One such ...

Conditions Affecting Social Space in Drosophila melanogaster

Conditions Affecting Social Space in Drosophila melanogaster

在条件影响社会空间<em&gt;果蝇</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

High Throughput Assay to Examine Egg-Laying Preferences of Individual Drosophila melanogaster

高通量检测，以检查个体产蛋首<em&gt;果蝇</em

Recently, egg-laying preference of Drosophila has emerged as a genetically tractable model to study the neural basis of simple decision-making processes. ...

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

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

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

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

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

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 ...

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

Monitoring Cell-to-cell Transmission of Prion-like Protein Aggregates in Drosophila Melanogaster

监测果蝇中朊病毒样蛋白聚集物的细胞间传输

Protein aggregation is a central feature of most neurodegenerative diseases, including Alzheimer&#39;s disease (AD), Parkinson&#39;s disease (PD), ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...