感谢Ricardo Dolmetsch博士（斯坦福大学医学院;现任隶属机构:诺华生物医学研究所），协助开展此项分析，马修·伍德博士，宝马爱原，以及董秀金博士对手稿的批判性阅读。

所有动物手术均经筑波大学动物护理与使用委员会（IACUC）批准进行。 解剖<ol><li>为了准备胚胎13-14日ICR或C57BL / 6胎儿，通过颈椎脱位安乐死怀孕的小鼠。取出子宫，并用汉克平衡盐溶液（HBSS）将其放入培养皿中。冲洗干净，以清除血液。 </li><li>使用镊子，将子宫转移到具有70％乙醇的新培养皿中以消毒。 </li><li>将子宫转移到带有HBSS的新培养皿中。使用消毒的解剖镊子和手术剪刀从子宫去除胎儿。 </li><li>使用外科剪刀切断胎儿。将它们放入带有HBSS的新培养皿中。 </li><li>取出皮肤和头骨，取出大脑使用灭菌解剖钳。将它们放入带有HBSS的培养皿中。 注意:从这一步开始，最好在干净的情况下进行该程序替补。 </li><li>在立体显微镜下使用灭菌解剖镊子取出脑膜。使用镊子切除基底神经节，海马和小脑。使用弯尖钳，将皮质转移到带有HBSS的新培养皿中。 </li><li>使用一次性滴管或25 mL移液管收集所有皮质。将其放入15 mL管中，然后用移液器取出HBSS。 </li><li>将3mL脑皮质酶溶液加入到含有皮质的管中。将该管放入水浴中，37℃下孵育5分钟。 注意:用0.25％胰蛋白酶和1mM EDTA制备由磷酸盐缓冲盐水（PBS）组成的脑皮质培养物。用0.22μm过滤器灭菌。 </li><li>在含有皮质的管中再加入3 mL脑皮质酶溶液，并在37°C的水浴中孵育另外5 min。 </li><li>使用5 mL移液管取出脑皮质酶溶液，加入5 mL Dulbe含有10％胎牛血清（FBS）的cco&#39;s Modified Eagle Medium（DMEM）。 </li><li>通过使用无菌P1000提示的5 mL移液管轻轻上下移动皮质分离皮质。再次使用带有P200尖端的5 mL移液管移液。 </li><li>将40μm尼龙细胞过滤器放在50mL管上，过滤悬浮液以除去碎屑。 </li><li>在室温下以420×g离心5分钟，然后除去DMEM培养基。 </li><li>加入5 mL脑皮层培养液，轻轻重悬细胞沉淀。 注意:准备一个脑皮层培养基，由补充有1×B-27补充剂，2mM L-谷氨酰胺和100U / mL青霉素链霉素的神经元基础培养基组成。 </li><li>使用血细胞计数器计数细胞，并使用脑皮层培养基调节细胞浓度（根据需要）。 </li><li>平板3.0×10 6皮质神经元，每个涂覆的35毫米玻璃底盘2.0毫升。 注意:电镀前，涂上具有0.1mg / mL聚-D-赖氨酸氢溴酸盐（PDL）或0.01％聚-L-鸟氨酸（PLO）的35mmm玻璃底盘。在37℃下孵育3小时-O / N，并用PBS洗涤至少3次。 </li></ol>成像囊泡运动<ol><li>准备标记每种类型囊泡的质粒。例如，使用EGFP-Rab5用于早期内体，EGFP-Rab7用于晚期内含体，LAMP-EGFP用于溶酶体，EGFP-LC3用于自噬体8,13。 注意:这些质粒可根据tsuruta.fuminori.fn@u.tsukuba.ac.jp的要求提供。通常使用转染试剂可以用这些质粒转染3〜5天体外 （DIV）神经元。 </li><li>通过移液将200μg无血清培养基混合4.0μg质粒。在单独的管中，在200μL无血清培养基中稀释8μL转染试剂（参见表格 ）。在室温下孵育5分钟。 </li><li>通过移液将步骤2.2中的质粒溶液和转染剂溶液混合。在室温下孵育20分钟。 </li><li>将无血清培养基和400μL来自步骤2.3的质粒溶液加入到每个涂覆的玻璃底盘中。在37℃下在5％CO 2培养箱中孵育30分钟。用2 mL大脑皮质培养基代替培养基。在37℃下在5％CO 2培养箱中孵育神经元1-2天。 </li><li>转染1 - 2 d后，选择转染的细胞进行图像采集。 注意:小于7 DIV的早产神经元具有呈现动态囊泡运动的趋势。选择适度表达荧光探针的神经元，因为当选择高度表达的神经元时，无法获得清晰的图像。此外，选择具有长轴突和复杂树突的典型神经元形状，例如锥体神经元，以便于鉴定轴突和树突（参见图1A）。 </li><li>使用延时成像系统进行图像采集: 对于成像，请使用配备有40X Plan Apo 0.95NA或100X Plan Apo VC NA1.4物镜的电荷耦合器件（CCD）相机的荧光显微镜。使用孵育系统控制温度在37°C。在图像采集软件控制的100s周期内以1帧/ 5s获取神经元图像。或者，以更短的间隔和更长的时间段采集图像，例如在300s的时间段内每2秒一帧。 注意:各种应用程序可用于拍摄顺序图像，其中许多应用将适用于此方法。因此，没有特别的应用推荐;相反，每个研究者都应该使用任何可用的应用程序。 </li></ol>图像分析<ol><li>使用手动跟踪14在ImageJ软件中打开所有顺序图像。 注意:到目前为止，手动跟踪一直是wi用于跟踪实验15,16,17,18,19。手动跟踪由FabriceCordelières博士研发。详细的书面说明可在线20 。为了分析，利用足够数量的图像来提高成像数据的质量。建议使用20张以上的图像。 </li><li>要组合顺序图像，请选择&lt;图像&gt;→&lt;堆叠&gt;→&lt;图像到堆叠&gt;。单击&lt;确定&gt;。 </li><li>选择&lt;插件&gt;→&lt;手动跟踪&gt;;将弹出跟踪窗口。 </li><li>单击&lt;显示参数？&gt;复选框，并在参数部分中定义跟踪参数。 注意:可以设置几个参数，包括时间间隔，x / y校准，z校准，搜索中心的正方形尺寸点数，点大小，行宽和字体大小。在这个简化的测定中，定义了时间间隔和x / y校准。在其他实验情况下，也可能需要定义其他参数。这里使用的参数如下:&lt;时间间隔&gt;为5秒，对于40X Plan Apo 0.95NA物镜，&lt;x / y校准&gt;为0.26642μm，对于100X Plan Apo VC NA1.4为0.10657μm物镜。 </li><li>定义参数后，单击&lt;添加轨迹&gt;开始新的轨道。 </li><li>在确定感兴趣的囊泡（ 例如， 图1A中的蓝色和红色箭头所示的囊泡（右图））后，点击顺序图像中信号的中心记录xy坐标。记录的xy坐标，距离和速度的结果显示在新窗口中。 注意:在溶酶体的情况下，大的明亮的荧光信号（ 例如，ure 1A（右图），橙色箭头）经常在神经元中观察到。由于这些信号偶尔表现出积聚的囊泡或病理性静脉曲张，因此应避免这些信号以量化运动性。 </li><li>重复步骤3.6从所有顺序图像收集囊泡的xy坐标;在选择参考点后，每个图像自动前进到连续图像。 </li><li>将这些数据导出到新表（ 例如电子表格）。使用适当的软件创建一个合适的图表（参见表格材料 ）。 注意:要分析数据，选择&lt;距离&gt;列，总结该列的所有值，并将运动距离显示为条形图， 如图1B所示 。此外，重复此步骤，计算总运动距离的平均值，并显示为条形图， 如图1C所示 。创建条形图和波形数据的软件如图所示材料表。 </li></ol>

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作者没有什么可以披露的。

该协议介绍了囊泡运动量的定量程序。在原代神经元中，内含体和溶酶体往往在年轻神经元（4-6 DIV）中表现出较高的运动能力。鉴于神经元必须将一些成分递送到前端以延长神经过程，因此膜运输应在此阶段动态发生。因此，使用年轻神经元观察神经元树突中的动态运动是重要的。此外，即使在年轻的神经元中，较大的囊泡也不会表现出较高的运动性。囊泡大小的调节可能涉及融合裂变步骤的频...

内源 - 溶酶体运输的精确控制对于调节神经元功能是必不可少的。值得注意的是，这些囊泡的动态运动是神经元形态，发育和生存调节的关键因素。该系统的缺陷引起严重的神经元疾病1,2 。将囊泡运输与神经元疾病相关联的分子机制被认为是复杂的，并且有几个组织试图检查这种相关性。例如，已经报道，扰动的晚期内体运动性与尼曼 - 皮克C病3 （由溶酶体缺陷引起的遗传性神经变性疾病）显着相关。另一个例子是溶酶体Ca 2+通道的突变， trpml1 ，其损害溶酶体运动，导致溶酶体贮积病4,5 ， </sup&gt; 6 。我们的研究小组报道，PtdIns（3,5）P 2转换的失调抑制了神经元中的内体和溶酶体运动，导致应激反应脆弱性增加7,8 。 PtdIns（3,5）P 2的代谢调节，其主要定位于晚期内体和溶酶体上，在多种细胞功能中起重要作用，包括囊泡运输和融合裂变过程9,10 。由于受损的PtdIns（3,5）P 2周转导致严重的神经变性11,12 ，内体 - 溶酶体运动的异常调节可能是了解神经变性发病机理的关键因素。对囊泡运动基础的分子机制的调查可能因此提供有希望的线索，可以加深我们的不便了解几种神经元疾病。 在本文中，我们引入了一种有价值的方法来量化神经元中的囊泡运动，使用自由软件包称为手动跟踪。目的是开发一种快速量化方法来分析囊泡运动。该定量是通过点击延时电影的每个帧中的参考点的标准方法进行的。与其他应用程序不同，使用手动跟踪软件使此方法非常简单，具有广泛的实用性。此外，该方法也适用于其他细胞，例如胶质细胞。虽然这种方法是原始的，但它可以应用于各种分析，包括细胞运动和形态变化。例如，在跨越图像序列定义参考点之后，可以使用数据分析和图像处理软件从连续图像中提取关于参考点的位置和每个位置处的时间的信息洁具。综合起来，这种方法简单而有力，有助于提高基于膜运输的研究的效率的发展，例如检查内体 - 溶酶体功能的研究。

<table><tbody><tr><td>Neurobasal medium</td><td>Thermo Fisher</td><td>21103-049</td><td></td></tr><tr><td>Dulbecco&#039;s modified eagle medium (DMEM)</td><td>Wako</td><td>044-29765</td><td></td></tr><tr><td>Opti-MEM</td><td>Thermo Fisher</td><td>31985070</td><td>Serum free medium</td></tr><tr><td>Hank&#039;s balanced salt solution (HBSS)</td><td>Thermo Fisher</td><td>14170112</td><td></td></tr><tr><td>Penicilin Streptmycin</td><td>Thermo Fisher</td><td>15140122</td><td></td></tr><tr><td>L-Glutamine</td><td>Thermo Fisher</td><td>25030081</td><td></td></tr><tr><td>B-27 Supplements</td><td>Thermo Fisher</td><td>17504044</td><td></td></tr><tr><td>2.5% Trypsine</td><td>Thermo Fisher</td><td>15090046</td><td></td></tr><tr><td>Poly-D-Lysine hydrobromide (PDL)</td><td>Sigma</td><td>P7280</td><td></td></tr><tr><td>Poly-L-Ornithine (PLO)</td><td>Sigma</td><td>P4957</td><td></td></tr><tr><td>Nylon cell strainer</td><td>Corning</td><td>431750</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher</td><td>11668027</td><td>Transfection reagent</td></tr><tr><td>Polyethyleneimine &quot;Max&quot; (PEI)</td><td>polysciences</td><td>24765</td><td>Transfection reagent. As an alternative to Lipofectamine2000, 0.1mg/mL Polyethyleneimine dissolved in sterilized water is available. But it is low efficiency and high toxicity.</td></tr><tr><td>BIOREVO BZ-9000</td><td>Keyence</td><td>NA</td><td></td></tr><tr><td>Incubation system INUG2-K13</td><td>Tokay Hit</td><td>NA</td><td></td></tr><tr><td>GraphPad Prism version 6.0</td><td>GraphPad Software</td><td>NA</td><td></td></tr><tr><td>Excel version 15</td><td>Microsoft</td><td>NA</td><td></td></tr><tr><td>ImageJ verion 1.47</td><td>NA</td><td>NA</td><td></td></tr></tbody></table>

quantification of endosome and lysosome motilities in cultured neurons using fluorescent probes

在大脑中，膜运输系统在调节神经元功能，如神经元形态，突触可塑性，存活和胶质细胞通讯中发挥重要作用。迄今为止，许多研究报道，这些系统中的缺陷引起各种神经元疾病。因此，了解囊泡动力学的机制可能会提供有助于治疗几种神经元疾病的有影响力的线索。在这里，我们使用ImageJ平台的软件插件描述了一种量化囊泡运动的方法，例如运动距离和运动速度。为了获得定量图像，我们用EGFP标记的囊泡标记蛋白标记神经元内体 - 溶酶体结构，并使用延时显微镜观察囊泡的运动。该方法非常有用，简化了神经突，如轴突和树突，以及神经元和神经胶质细胞的神经元测量囊泡运动。 Furthermo这种方法可以应用于其他细胞系，如成纤维细胞和内皮细胞。这种方法可以为我们对膜运输的理解提供有价值的推动。

该测定设计用于测量体外培养中的囊泡动力学。该测定用于确定与神经元形态和存活相关的囊泡运动性。 图1A和B显示了显示神经元溶酶体运动性的代表性数据。皮质神经元用溶酶体标记LAMP-EGFP转染，并使用标准荧光显微镜观察。以前曾报道，即使在静息神经元中，特异性内体溶酶体囊泡也是高度移动的。实际上，这些结果表明在相同的枝?...

Watch this Scientific Journal Video about Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes at JoVE.com

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

膜运输的调查对于了解神经元功能至关重要。在这里，我们介绍一种量化神经元囊泡运动性的方法。这是一种方便的方法，可以适应于神经系统膜运输的量化。

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

quantification-endosome-lysosome-motilities-cultured-neurons-using

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

8.0K 次观看.  University of Tsukuba. 膜运输的调查对于了解神经元功能至关重要。在这里，我们介绍一种量化神经元囊泡运动性的方法。这是一种方便的方法，可以适应于神经系统膜运输的量化。 

视频: Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

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 ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

表征分子马达的组成对运动轴突货物用"货物映射"分析

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

TIRFM和pH敏感的GFP-探针来评估神经递质囊泡动力学SH-SY5Y神经母细胞瘤:细胞成像和数据分析

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

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 ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

细胞器运输的培养<em&gt;果蝇</em&gt;细胞：S2细胞系和原代神经元。

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

生物学

Real-Time Fluorescent Measurement of Synaptic Functions in Models of Amyotrophic Lateral Sclerosis

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe dementia (FTLD) is dysfunction of communication between neurons and motor neurons and muscle. The underlying process of synaptic transmission involves membrane depolarization-dependent synaptic vesicle fusion and the release of neurotransmitters into the synapse. This process occurs through localized calcium influx into the presynaptic terminals where synaptic vesicles reside. Here, the protocol describes fluorescence-based live-imaging methodologies that reliably report depolarization-mediated synaptic vesicle exocytosis and presynaptic terminal calcium influx dynamics in cultured neurons.
Using a styryl dye that is incorporated into synaptic vesicle membranes, the synaptic vesicle release is elucidated. On the other hand, to study calcium entry, Gcamp6m is used, a genetically encoded fluorescent reporter. We employ high potassium chloride-mediated depolarization to mimic neuronal activity. To quantify synaptic vesicle exocytosis unambiguously, we measure the loss of normalized styryl dye fluorescence as a function of time. Under similar stimulation conditions, in the case of calcium influx, Gcamp6m fluorescence increases. Normalization and quantification of this fluorescence change are performed in a similar manner to the styryl dye protocol. These methods can be multiplexed with transfection-based overexpression of fluorescently tagged mutant proteins. These protocols have been extensively used to study synaptic dysfunction in models of FUS-ALS and C9ORF72-ALS, utilizing primary rodent cortical and motor neurons. These protocols easily allow for rapid screening of compounds that may improve neuronal communication. As such, these methods are valuable not only for the study of ALS but for all areas of neurodegenerative and developmental neuroscience research.

Two related methods are described to visualize subcellular events required for synaptic transmission. These protocols enable the real-time monitoring of the dynamics of presynaptic calcium influx and synaptic vesicle membrane fusion using live-cell imaging of in vitro cultured neurons.

肌萎缩性侧索硬化症模型中突触功能的实时荧光测量

Before neuronal degeneration, the cause of motor and cognitive deficits in patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal lobe ...

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient bidirectional transport by motor proteins. Various reports have suggested that defects in axonal transport are associated with neurodegenerative diseases. Also, the mechanism of the coordination of multiple motor proteins has been an attractive topic. Since the axon has uni-directional microtubules, it is easier to determine which motor proteins are involved in the movement. Therefore, understanding the mechanisms underlying the transport of axonal cargo is crucial for uncovering the molecular mechanism of neurodegenerative diseases and the regulation of motor proteins. Here, we introduce the entire process of axonal transport analysis, including the culturing of mouse primary cortical neurons, transfection of plasmids encoding cargo proteins, and directional and velocity analyses without the effect of pauses. Furthermore, the open-access software "KYMOMAKER" is introduced, which enables the generation of a kymograph to highlight transport traces according to their direction and allow easier visualization of axonal transport.

The present protocol describes the whole procedure of analysis for axonal transport. In particular, the calculation of transport velocity, not including the pausing, and the visualization method using open-access software "KYMOMAKER" are shown here.

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient ...

<meta charset="utf8"></head><body>使用 GFP 标记的 RAB-3 增强活 秀丽隐杆线虫 的轴突转运分析

Optimizing Visualization of Axonal Transport of Endogenous Cargo by Fluorescence Microscopy in Living Caenorhabditis elegans

Axonal transport is a prerequisite to deliver axonal proteins from their site of synthesis in the neuronal cell body to their destination in the axon. Consequently, loss of axonal transport impairs neuronal growth and function. Studying axonal transport therefore improves our understanding of neuronal cell biology. With recent improvements in CRISPR Cas9 genome editing, endogenous labeling of axonal cargos has become accessible, enabling to move beyond ectopic expression-based visualization of transport. However, endogenous labeling often comes at the cost of low signal intensity and necessitates optimization strategies to obtain robust data. Here, we describe a protocol to optimize the visualization of axonal transport by discussing acquisition parameters and a bleaching approach to improve the signal of endogenous labeled cargo over diffuse cytoplasmic background. We apply our protocol to optimize the visualization of synaptic vesicle precursors (SVPs) labeled by green fluorescent protein (GFP)-tagged RAB-3 to highlight how fine-tuning acquisition parameters can improve the analysis of endogenously labeled axonal cargo in Caenorhabditis elegans (C. elegans).

<meta charset="utf8"></head><body>作者聚焦:可视化内源性轴突传输动力学的高级技术

The paper describes the optimization of fluorescence microscopy acquisition parameters to visualize the axonal transport of endogenous labeled cargos at single-neuron resolution in a living nematode.

通过荧光显微镜优化活体秀丽隐杆线虫中内源性货物轴突运输的可视化

Axonal transport is a prerequisite to deliver axonal proteins from their site of synthesis in the neuronal cell body to their destination in the axon. ...

使用荧光探针定量培养的神经元中的内体和溶酶体运动

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