Name: studying synaptic vesicle pools using photoconversion of styryl dyes
Uploaded: 2010-02-15T17:00:00
Description: Watch this Scientific Journal Video about Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes at JoVE.com

1）准备果蝇神经肌肉交界处（NMJ） <ol><li>准备标准果蝇生理盐水（氯化钙2 2毫米，130毫米，36毫米蔗糖氯化钠，氯化钾5毫米，2毫米氯化镁2，5毫米HEPES，pH值7.3 4。 </li><li>解剖生理盐水（1.1）的准备工作。果蝇幼虫背侧是不眠不休在Sylgard菜;背侧纵向切片，和内部器官将被删除。然后拉伸和不眠不休的准备工作。然后可以使用几个腹侧肌肉。 </li></ol><p class="jove_titl...

<ol>
	<li>Betz, W. J., Bewick, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+analysis+of+synaptic+vesicle+recycling+at+the+frog+neuromuscular+junction.">Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.</a> Science. 255, 200-203 (1992).</li><li>Henkel, A. W., L&#252;bke, J., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM1-43+dye+ultrastructural+localization+in+and+release+from+frog+motor+nerve+terminals.">FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.</a> Proc Natl Acad Sci USA. 93, 1918-1923 (1996).</li><li>Sandell, J. H., Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoconversion+of+some+fluorescent+markers+to+a+diaminobenzidine+product.">Photoconversion of some fluorescent markers to a diaminobenzidine product.</a> J Histochem Cytochem. 36, 555-559 (1988).</li><li>Kuromi, H., Kidokoro, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+optically+determined+size+of+exo/endo+cycling+vesicle+pool+correlates+with+the+quantal+content+at+the+neuromuscular+junction+of+Drosophila+larvae.">The optically determined size of exo/endo cycling vesicle pool correlates with the quantal content at the neuromuscular junction of Drosophila larvae.</a> J Neurosci. 19, 1557-1565 (1999).</li><li>Denker, A., Kr&#246;hnert, K., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revisiting+synaptic+vesicle+pool+localization+in+the+Drosophila+neuromuscular+junction.">Revisiting synaptic vesicle pool localization in the Drosophila neuromuscular junction.</a> J Physiol (Lond). 587, 2919-2926 (2009).</li><li>Rizzoli, S. O., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+organization+of+the+readily+releasable+pool+of+synaptic+vesicles.">The structural organization of the readily releasable pool of synaptic vesicles.</a> Science. 303, 2037-2039 (2004).</li><li>Darcy, K. J., Staras, K., Collinson, L. M., Goda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+sharing+of+recycling+synaptic+vesicles+between+presynaptic+boutons.">Constitutive sharing of recycling synaptic vesicles between presynaptic boutons.</a> Nat Neurosci. 9, 315-321 (2006).</li><li>Harata, N., Ryan, T. A., Smith, S. J., Buchanan, J., Tsien, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+recycling+synaptic+vesicles+in+hippocampal+neurons+by+FM1-43+photoconversion.">Visualizing recycling synaptic vesicles in hippocampal neurons by FM1-43 photoconversion.</a> Proc Natl Acad Sci USA. 98, 12748-12753 (2001).</li><li>Lange, R. P. J. d. e., de Roos, A. D. G., Borst, J. G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+modes+of+vesicle+recycling+in+the+rat+calyx+of+Held.">Two modes of vesicle recycling in the rat calyx of Held.</a> J Neurosci. 23, 10164-10173 (2003).</li><li>Richards, D. A., Guatimosim, C., Rizzoli, S. O., Betz, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+pools+at+the+frog+neuromuscular+junction.">Synaptic vesicle pools at the frog neuromuscular junction.</a> Neuron. 39, 529-541 (2003).</li></ol>

应考虑几个关键步骤： <ul><li>民建联的潜伏期后，才应彻底清洗和淬火的准备。否则非反应的戊二醛将与民建联，并导致其降水（通常在平面晶体的形式，这是不是电子致密）。这种沉淀大量发生的筹备工作是很少用于电子显微镜使用。 </li><li>照明时间应优化，测试与几个不同的光照时间相同的筹备工作。根据我们的经验，实现最佳的转换（图2B）在5分钟左右的时间窗口（例如，35至照明开始...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>FM 1-43</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>F10317</td>
<td>&nbsp;</td>
<tr>
<td>Epon resin</td>
<td>&nbsp;</td>
<td>Plano</td>
<td>R1030</td>
<td>&nbsp;</td>
<tr>
<td>di-aminobenzidine hydrochloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D5905</td>
<td>&nbsp;</td>
<tr>
<td>50% Glutaraldehyde</td>
<td>&nbsp;</td>
<td>AppliChem</td>
<td>A3166</td>
<td>EM grade</td>
</tr>
<tr>
<td>Sylgard</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>104186298</td>
<td>&nbsp;</td>
<tr>
<td>Axioskop 2 FS plus</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Objective 20x 0.5 NA</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>Dry objective</td>
</tr>
<tr>
<td>100W Hg Lamp</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Lamp housing with back mirror</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>1007-980</td>
<td>&nbsp;</td>
<tr>
<td>MRm camera</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>0445-554</td>
<td>Image acquisition</td>
</tr>
<tr>
<td>Ex. Filter (HQ 470/40)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F49-671</td>
<td>&nbsp;</td>
<tr>
<td>Dichroic (495 DCLP)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F33-100</td>
<td>&nbsp;</td>
<tr>
<td>Em. Filter (HQ 500 LP)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F42-018</td>
<td>&nbsp;</td>
<tr>
<td>EM</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Proscan CCD HSS</td>
<td>&nbsp;</td>
<td>Proscan Electronic Sys.</td>
<td>&nbsp;</td>
<td>1024 x 1024</td>
</tr>		</tbody>
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studying synaptic vesicle pools using photoconversion of styryl dyes

突触囊泡与质膜（胞吐）的融合是一种神经递质的释放和神经元沟通的必要步骤。囊泡，然后从质膜（内吞作用）检索与一般泳池内的神经末梢的囊泡和分组，直到他们接受一个新的外和内吞作用周期（囊泡循环）。这些过程进行了研究，利用各种技术，如电子显微镜，电录音，安培和电容测量。更重要的是，在过去二十年了荧光标记的标记出现，使光学技术囊泡回收动态跟踪。最常用的标记之一是苯乙烯或FM 染料 1;结构，所有的FM染料含有亲水性的头部和亲油性的尾部通过芳香环和一个或多个双键（图1B）连接。一个古典调频染料实验，以标签的小泡池组成，在洗浴过程中的神经刺激的准备（图1Ai）（电动或高 K +）与染料。这种诱导囊泡循环和随后的染料加载到最近内吞囊泡（图1A I - III）。用染料，染料免费洗澡的刺激第二轮装载水泡后会触发通过胞吐FM释放（图1A IV - V），可以通过监测荧光强度降低（脱色）的过程。 虽然调频染料囊泡循环领域作出了巨大贡献，它是不可能的，以确定使用传统的荧光显微镜下精确定位或个别囊泡形态。出于这个原因，我们在这里解释调频染料，还可以通过光转化为使用电子显微镜吞标记，。光转化技术，利用荧光染料的财产，以强烈的光照下产生的活性氧物种。荧光标记的准备工作都淹没在含有二氨基联苯胺（DAB）解决方案和照明。染料分子产生活性物种的氧化，形成一个稳定的，不溶性沉淀物，有一个黑暗的外观，可以很容易地在电子显微镜2,3杰出民建联。正如民建联只在紧邻的荧光分子（如活性氧是短命）的氧化，该技术确保只荧光标记的结构是将含有电子致密沉淀。因此，该技术允许积极回收细胞器的确切位置和形态的研究。

Watch this Scientific Journal Video about Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes at JoVE.com

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

调频染料已宝贵的帮助，在突触动力学的理解。外长荧光显微镜下通常遵循在不同的刺激条件下。然而，与电子显微镜相结合的FM染料的光转化允许不同的突触小泡池的可视化，以及其他超微结构组件，在突触boutons。

突触小泡池，利用苯乙烯染料的光转化的研究

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Research

JoVE Journal

Neuroscience

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

科研

神经科学

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

<sub&gt; 1</sub&gt; F<sub&gt; O</subATP酶微囊的制备和表演膜片钳记录的Submitochondrial泡膜技术

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

使用调频染料诱发过程中，自发的，微型突触活动检查突触囊泡循环的

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

星形细胞转运电流派生谷氨酸间隙的时间进程与反卷积分析

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

突触质膜和突触后密度蛋白采用不连续蔗糖梯度准备

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

海马神经元中突触囊泡吞的测量

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

Culturing In Vivo-like Murine Astrocytes Using the Fast, Simple, and Inexpensive AWESAM Protocol

使用快速、简单且便宜的 AWESAM 协议培养在体内像小鼠星形胶质细胞

The AWESAM (a low-cost easy stellate astrocyte method) protocol entails a fast, simple, and inexpensive way to generate large quantities of in vivo-like ...

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 ...

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Overexpressing 突触前蛋白培养神经元突触囊泡再生的光学检测

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...