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> 2）刺激和FM染色<ol><li>可取的做法是在光线不足的条件下做的所有下列步骤，以保护漂白调频染料。 </li><li>化学刺激的神经，含钾高的缓冲区使用。准备果蝇生理盐水（见1.1），含氯化钾90毫米和10 FM1 -​​ 43染料微米。保持避光的解决方案。 </li><li>与先前准备的缓冲1分钟，在室温下沐浴的准备。 </li><li>与标准果蝇生理盐水（1.1）洗删除外，FM1 -​​ 43染料。迅速进行固定。 </li></ol> 3）固定<ol><li>巴斯准备在磷酸盐缓冲液（PBS）用2.5％戊二醛在室温为45分钟。形态特征的保存好，以多聚甲醛戊二醛是首选。 </li><li>用PBS洗一次，然后离开NH 4 CL 100毫米淹没在15分钟的准备。这一步是为了解渴剩余的戊二醛固定液的自由醛基。 </li><li>正常PBS洗的NH 4 CL解决方案。 </li></ol> 4）光转化<ol><li> NMJ准备孵育30分钟，在4 °彗星在PBS含1.5毫克的二氨基联苯胺（DAB）ML - 1。 </li><li>荧光显微镜下样品的位置。查找和集中使用相对低倍率水浸泡的目的（20X 0.5 NA）的荧光信号。 </li><li>照明灯强度最大的样本，直到FM染料是完全漂白（图1C）。要控制，如果光转化有发生，建议在透射光下检查样品前后FM染料脱色一步。如果光转化发生，深褐色沉淀预期（图1C）。光照时间是可变的，取决于后的光照强度，并呼吁民建联渗透进入准备也。对于大多数准备工作，我们发现〜30-45分钟的光照时间是最佳的，当使用20倍的目标。较短的光照时间用于更高的放大倍率目标（60X）。 </li><li>用于照明，我们更愿意使用水银灯泡，一盏灯，一回镜子，收集背散射光束，因此增加总强度的住房。 </li></ol> 5）处理EM样本<ol><li> Osmication <ol class="lalpha"><li>准备一个体积水树量的四氧化锇每个样品（约300μL）的解决方案。使用四氧化锇必须做的引擎盖下，使用手套和赞成保护设备的工作。 </li><li>孵育锇酸溶液在室温（引擎盖下）1小时的准备。 </li><li>广泛洗净用PBS（4-5倍，5分钟），每个样品转移到一个干净的玻璃瓶中。 </li></ol></li><li>脱水<ol class="lalpha"><li>准备单独的解决方案，包含30，50，70，90，95和100％的乙醇。 </li><li>每个样品中加入1毫升30％乙醇5分钟。 </li><li> 1毫升50％的乙醇添加到每个样品5分钟。 </li><li> 5分钟，每个样品中加入1毫升70％的乙醇。 </li><li> 5分钟，每个样品中加入1毫升90％的乙醇。 </li><li> 5分钟1毫升95％的乙醇添加到每个样品重复3倍。 </li><li> 5分钟1毫升100％的乙醇添加到每个样品重复3倍。 </li></ol></li><li>嵌入和进一步处理<ol class="lalpha"><li> 1体积EPON树脂混合乙醇量。将它添加到编制下连续旋转（2-4小时）。 </li><li>放置在100％EPON开放烧瓶的准备，让剩余的乙醇蒸发（4-6小时）。 </li><li>在60℃放置36小时在模具的准备。 </li><li>电子显微镜处理。工艺编制在50-80纳米的薄片，用标准的超薄切片程序。 </li></ol></li><li>电子显微镜成像<ol class="lalpha"><li>影像使用传统的电磁程序的准备工作。 </li></ol></li></ol> 6）代表性的成果图1C和2概述了预期的成果。在照明人权法的形成过程结果WN民建联的沉淀物，这将是在荧光和传输成像可见。第一次出现是在照明的FM染料染色的荧光消失。醛固色剂引起的背景荧光，相反，将在整个实验过程中可见。漂白〜10-20分钟的地方，通常后的照明开始。没有光转化产品通常可以观察到在这个时间点。 照度应继续〜10分钟后的筹备工作会变成棕色遮阳由于民建联的积累（图1C四），目前还不清楚为什么民建联降水和FM染料褪色不同时发生，这是一个比较大的氧化民建联量需要积累前聚集和沉淀，因此，这种反应是比漂白过程慢。然而，在这个阶段，准备不准备电子显微镜处理，如民建联的降水可能是不完整的。我们宁愿等待5-10分钟的时间，在这期间准备（突触前神经终端）承担了深褐色（或黑色）的颜色，完成转换的指示。可以观察到在这个阶段的编制一般表面有轻微的转换（例如，在神经肌肉交界处的肌纤维）。它是最有可能涉及到的民建联诱导自体荧光（和/或固定液荧光）的转换，而且它不是有害的程序。 电子显微镜的准备工作，然后处理，并应检查存在暗（标记）囊泡。正如我们在5,6证明，这些囊泡比非标记的多密集，因此很容易区别开来（图2B ）。为了增加以及区分不同类型的囊泡的机会应该是执行，没有对比增强后的部分染色（无醋酸铀或柠檬酸铅染色）;锇染色足以观察到大多数的细胞成分，而不是作为民建联沉淀黑暗。 <img src="/files/ftp_upload/1790/1790fig1.jpg" alt="图1" /> 图1：FM染料和光转化为使用 。 （a）代表一个用于装载和distaining囊泡的刺激下使用调频染料的经典实验的步骤。 （ 一 ）在FM染料孵育样品含有缓冲区。 （ 二 ）电或化学刺激（）来诱导调频染料存在囊泡融合。 （ 三 ）刺激后的序列的内吞作用将负载与FM染料仍然在细胞外的缓冲区目前一些水泡。 （ 四 ）更换外FM染料，用缓冲液冲洗。 （五）新的刺激会诱发加载囊泡胞吐作用释放后，他们的调频染料。 （二）示意图显示头部，桥梁和FM1 - 43染料的尾巴。 （三）在果蝇 NMJ的光转化实验的例子。 （ 一 ）加载后FM1 - 43清洗外部的染料分子和固定，NMJ，荧光的神经终端，可以观察到与传统epifluorescence的显微镜（63X ）。 （II - III）在连续照明的染料是完全漂白。 （ 四 ）当照明持续下去，会出现一个黑色由于二氨基联苯胺沉淀。比例尺代表10微米。 <img src="/files/ftp_upload/1790/1790fig2.jpg" alt="图2" /> 图2：EM photoconverted样品的例子（ 一 ）图像显示了神经末梢突触囊泡，但他们不photoconverted;这可能是没有足够的照明，或在组织中的差二氨基渗透造成的。 （二）与几个暗（填写）囊泡通过调频光转化突触布顿。 （三）以上的照明结果在整体暗的终端，没有水疱或细胞器是区分。比例尺代表200纳米。

<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>Catalogue Number</th>
		<th>Comment</th>
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<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>
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<td>Ex. Filter (HQ 470/40)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F49-671</td>
<td>&nbsp;</td>
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<td>Dichroic (495 DCLP)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F33-100</td>
<td>&nbsp;</td>
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<td>Em. Filter (HQ 500 LP)</td>
<td>&nbsp;</td>
<td>AHF</td>
<td>F42-018</td>
<td>&nbsp;</td>
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<td>EM</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Proscan CCD HSS</td>
<td>&nbsp;</td>
<td>Proscan Electronic Sys.</td>
<td>&nbsp;</td>
<td>1024 x 1024</td>
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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 ...

studying-synaptic-vesicle-pools-using-photoconversion-of-styryl-dyes

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Neuroscience

11.6K 次观看.  European Neuroscience Institute Göttingen. 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。

视频: Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

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

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

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

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

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 ...

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

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

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

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

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 ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

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

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 mouse and rat astrocyte monocultures: Brain cells can be isolated from different brain regions, and after a week of cell culture, non-astrocytic cells are shaken off by placing the culture dishes on a shaker for 6 h in the incubator. The remaining astrocytes are then passaged into new plates with an astrocyte-specific medium (termed NB+H). NB+H contains low concentrations of heparin-binding EGF-like growth factor (HBEGF), which is used in place of serum in medium. After growing in NB+H, AWESAM astrocytes have a stellate morphology and feature fine processes. Moreover, these astrocytes have more in vivo-like gene expression than astrocytes generated by previously published methods. Ca2+ imaging, vesicle dynamics, and other events close to the membrane can thus be studied in the fine astrocytic processes in vitro, e.g., using live cell confocal or TIRF microscopy. Notably, AWESAM astrocytes also exhibit spontaneous Ca2+ signaling similar to astrocytes in vivo.

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

The AWESAM protocol described here is optimal for culturing murine astrocytes in isolation from other brain cells in a fast, simple, and inexpensive manner. AWESAM astrocytes exhibit spontaneous Ca2+ signaling, morphology, and gene expression profiles similar to astrocytes in vivo.

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 ...

Pentylenetetrazole-Induced Kindling Mouse Model

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high dose, whereas sequential injections of a subconvulsive dose have been used for the development of chemical kindling, an epilepsy model. A single low-dose injection of PTZ induces a mild seizure without convulsion. However, repetitive low-dose injections of PTZ decrease the threshold to evoke a convulsive seizure. Finally, continuous low-dose administration of PTZ induces a severe tonic-clonic seizure. This method is simple and widely applicable to investigate the pathophysiology of epilepsy, which is defined as a chronic disease that involves repetitive seizures. This chemical kindling protocol causes repetitive seizures in animals. With this method, vulnerability to PTZ-mediated seizures or the degree of aggravation of epileptic seizures was estimated. These advantages have led to the use of this method for screening anti-epileptic drugs and epilepsy-related genes. In addition, this method has been used to investigate neuronal damage after epileptic seizures because the histological changes observed in the brains of epileptic patients also appear in the brains of chemical-kindled animals. Thus, this protocol is useful for conveniently producing animal models of epilepsy.

This protocol describes a method of chemical kindling with pentylenetetrazole and provides a mouse model of epilepsy. This protocol can also be used to investigate vulnerability to seizure induction and pathogenesis after epileptic seizures in mice.

戊四氮诱导点燃小鼠模型

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

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

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) 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

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane proteins become exposed at the cell surface. One of these proteins is Synaptotagmin-1 (Syt1). An antibody directed against the luminal domain of Syt1, once added to the culture medium, is taken up during the exo-endocytotic cycle. This uptake is proportional to the amount of SV recycling and can be quantified through immunofluorescence. Here, we combine Syt1 antibody uptake with double transfection of cultured hippocampal neurons. This allows us to (1) localize presynaptic sites based on expression of recombinant presynaptic marker Synaptophysin, (2) determine their functionality using Syt1 uptake, and (3) characterize the targeting and effects of a protein of interest, GFP-Rogdi.

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

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 ...

Evaluation of Synaptic Multiplicity Using Whole-cell Patch-clamp Electrophysiology

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic multiplicity). Synaptic multiplicity is plastic and changes throughout development and in different physiological conditions, being an important determinant for the efficacy of synaptic transmission. Here, we outline experiments for estimating the degree of multiplicity of synapses terminating onto a given postsynaptic neuron using whole-cell patch clamp electrophysiology in acute brain slices. Specifically, voltage-clamp recording is used to compare the difference between the amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and miniature excitatory postsynaptic currents (mEPSCs). The theory behind this method is that afferent inputs that exhibit multiplicity will show large, action potential-dependent sEPSCs due to the synchronous release that occurs at each synaptic contact. In contrast, action potential-independent release (which is asynchronous) will generate smaller amplitude mEPSCs. This article outlines a set of experiments and analyses to characterize the existence of synaptic multiplicity and discusses the requirements and limitations of the technique. This technique can be applied to investigate how different behavioral, pharmacological or environmental interventions in vivo affect the organization of synaptic contacts in different brain areas.

Here, we present a protocol for evaluating the functional synaptic multiplicity using whole-cell patch clamp electrophysiology in acute brain slices.

全细胞膜片电生理方法对突触倍数的评价

In the central nervous system, a pair of neurons often form&#160;multiple synaptic contacts and/or functional neurotransmitter release sites (synaptic ...