This work was funded by NIH grants DA013137, DA031604, and HD043680. Partial support was provided by a NIH T32 training grant in Biomedical-Behavioral science.

所有动物方案进行了审查，并经动物护理和使用委员会在南卡罗来纳大学（保证号码:A3049-01）的批准。 1.低密度胚胎神经文化<ol><li> 初级皮层神经元文化的制备 <ol><li>解决方案: <ol><li>由5毫克多聚L-赖氨酸溶解在10毫升硼酸盐缓冲液制备聚-L-赖氨酸原液。 </li><li>制备通过在49毫升硼酸缓冲液稀释1毫升聚L-赖氨酸库存工作溶液。 </li><li>通过395毫升卫生署2 O开始，然后添加的所有成分（1.24克硼酸+1.9克硼砂）准备硼酸盐缓冲液，pH值8.4。为1M NaOH调节pH值至8.4。调整至400毫升，并引擎盖下的0.2微米尼龙滤网过滤。 </li><li>通过445毫升卫生署2 O开始，添加所有其他配料准备HBSS液，pH值7.2（50毫升10X HBSS股票+1.2克HEPES）。由1N盐酸，BRI调节pH至7.2纳克至500ml，并与发动机罩下的0.2微米尼龙滤网过滤。 </li><li>准备电镀培养基:DMEM / F12，用10％的胎牛血清。与引擎盖下的0.2微米的尼龙膜滤膜过滤。 </li><li>准备完全生长培养基:向50ml Neurobasal培养基中添加添加剂（500微升GLUTAMAX（100X）+ 500微升葡萄糖+1毫升B-27（50X）+ 500微升抗生素抗真菌溶液（100倍）+ 100微升7.5％小苏打与引擎盖下的0.2微米的尼龙膜滤膜过滤。 </li></ol></li><li>文化前两天: <ol><li>外套1235毫米玻璃底菜用2ml聚L-赖氨酸的工作溶液，并保持罩O / N下。 </li></ol></li><li>文化前一天: <ol><li>从培养皿空涂层剂并用的DDH 2 O冲洗允许碗碟干燥发动机机罩下一个小时。 </li><li>制备电镀培养基（DMEM / F12，用10％胎牛血清（总溶液的10％）。加入2毫升培养基成每道菜和孵育O / N 在 37℃，5％ 的 CO 2。 注意:培养时，为了避免塑料培养皿是很重要的。玻璃底菜（适用于倒置显微镜）或盖玻片（适用于正置显微镜）的使用产生锐利和清晰的图像。相比于塑料底皿中，在玻璃底培养皿荧光显微镜中避免了不必要的色调或眩光。 </li></ol></li></ol></li><li> 细胞培养协议: <ol><li>放冷藏的HBSS缓冲液（100-150毫升），100 25mm培养皿，镊子，剪刀，50ml试管，并在UV光下15ml试管中罩。 </li><li>安乐死有5％的七氟烷吸入致死孕鼠引擎盖下培养室之外。 </li><li>用70％乙醇，帐幕使用镊子而从尾部上腹部cavity.Open用剪刀以暴露胎盘子宫的中间切割下腹部的皮肤消毒。 </li><li>删除8-10 E18胎儿。解放军CE含HBSS液的培养皿8-10胎儿。 </li><li>握住钳子胎儿的后脑​​勺，然后用剪刀从身体切断头。将其放置在另一个培养皿中充满了5-7毫升的HBSS。转移菜罩。 </li><li>装满冷的HBSS两个额外百毫米培养皿。 </li><li>皮尔一边头骨和大脑舀到一个新的培养皿中充满了HBSS。 </li><li>用锋利弯钳，独立的小脑和脑干脑，大脑再冷HBSS中转移到一个新的培养皿。 </li><li>用镊子固定，半球用弯钳分离。删除脑膜;隔离额叶皮层和传输块放入标有15ml离心管中。 </li><li>用新鲜2毫升HBSS填充15ml试管中，加入20微升胰蛋白酶EDTA（10倍与0.5％胰蛋白酶和5.3毫米EDTA。浓缩的混合物），以每次15毫升管。 </li><li>孵育10-15分钟在室温。轻轻旋转管每隔几分钟，不允许沉降。 </li><li>房颤之三10-15分钟，用玻璃吸管移除旧的HBSS，并与新的HBSS冲洗两次。 </li><li>浸入胰蛋白酶抑制剂（1毫克/毫升，2毫克胰蛋白酶在2毫升HBSS抑制剂），拌匀，让它坐5分钟。用新鲜的HBSS洗两次。 </li><li>使用玻璃吸管与橡胶球，磨碎脑件10-15倍非常缓慢，以避免bubbles.Triturate再次使用校准的移液管和一个无菌尖端具有减小尖端直径非常缓慢直至均匀。 </li><li>转移解离细胞以所需密度预先制备涂布培养皿（50个细胞/平方毫米）。孵育O / N在37℃，5％CO2。 </li><li> 24小时后，用新鲜的完全生长的无血清Neurobasal培养基替代DMEM / F12培养基。 </li><li>保持在任何时候都将细胞在5％的CO 2/95％的室内空气加湿培养箱用于实验前至少12-14天。补充细胞每5-6天用新鲜的Neurobasal培养基更换旧培养基的约50％。 </li></ol></li></ol> 2.荧光标记和免疫组化注:原代皮质细胞培养物的免疫荧光标记是在玻璃底部35 MM细胞培养皿进行与1毫升工作体积。 <ol><li>用磷酸盐缓冲盐水pH值7.4（PBS）洗玻璃底皿两次。 </li><li>用4％多聚甲醛的细胞在室温15分钟。 </li><li>用PBS洗涤细胞两次并在PBS中与0.1％的Triton X-100通透5分钟。 </li><li>处理细胞在室温20分钟用F-肌动蛋白特异性染色，AlexaFluor 488鬼笔环肽（1:40，25微升鬼笔环肽的在1毫升PBS）中。 </li><li>冲洗细胞两次，用PBS，并在室温，用10％正常山羊血清阻断1-2小时。 </li><li>稀释的鸡多克隆抗MAP2抗体（1:2,500）在PBS中的2％正常山羊血清，并在4℃孵育O / N </li><li>温育后，冲洗在PBS两次。 </li><li>稀释二级抗体（Alexa的 - [RED 594缀合的山羊抗鸡IgG（1:500），用2％正常山羊血清和在室温下孵育2小时。 </li><li>用PBS冲洗，加10微升/赫司特染料菜。 </li><li>孵育3分钟在室温下，并用PBS洗两次。 注:现在标​​记的细胞准备第3步（F-actin的泪点计数）。 </li><li>保持细胞样品用100微升抗淬灭剂作为盖片剂，并保持在 4℃在黑暗中长时间存放。 注意:鬼笔环肽是在PBS中在长达1周和荧光能与抗褪色盖片的试剂保留在黑暗+4℃下相对稳定。不过，最佳的图像是从染色后1-3天获得自鬼笔环肽可以开始扩展存储扩散出去。 </li></ol> 3. F-肌动蛋白泪点计数<ol><li>打开荧光显微镜，并切换到20倍的目标。打开显微镜软件，并设置方案在1280×960像素的图像尺寸，以及0.1在1倍变焦7微米/像素的图像分辨率。 </li><li>根据获取绿色（495纳米）/红（613 nm）的荧光渠道共标记F-肌动蛋白/微管相关蛋白神经元的图像。 </li><li>选择5格林（F-肌动蛋白）/红（MAP2）免疫标记/蓝（赫司特）有明确界定的树突状乔木单个神经元的荧光图像。 </li><li>确定二阶树突段（长度范围25-75微米）连续MAP2免疫的F-肌动蛋白丰富的结构。 </li><li>图像的所选区域旋转到水平位置。复制和粘贴为新的图像。 </li><li>减去的图像的背景，使用为每个菜一致的调整。 </li><li>算上亮绿色F-肌动蛋白泪点和手动由训练有素的独立观察员跟踪选定的树突状段的长度。将数据导出到电子表格文件。 </li><li>计算由长度（L）的MAP2标记树突将总F-肌动蛋白标记的泪点（N）的密度。 Express数据为F-AC的数每10微米枝蔓锡泪点 注意:泪点F-肌动蛋白荧光与在树突轴染色的平均强度以上的至少50％的峰强度的（大小≤1.5微米）包括在每个所选树突段。 </li></ol>

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None of the authors have conflicts of interest to declare.

在这个协议中，我们描述了在35毫米的玻璃底菜低密度这使我们能够识别单个神经元树突培养大鼠皮层神经元。接下来，我们使用鬼笔环肽和MAP2染色检测树枝状的改变。然后，我们使用专门的软件来量化在F-肌动蛋白泪点的变化。 为了确定F-肌动蛋白泪点的个体神经元的神经全网络必须是清晰可见的变化，这使得从单个神经元适当二阶树突段的选择。低密度电镀是为了既可视?...

本研究的主要目标是开发测量（估计）的神经元的树突状网络的突触完整性的可靠方法。在这里，我们描述了使用鬼笔环肽染色和使用专门的（NIS元素）软件与后续分析树突免疫细胞化学（ICC）检测结合原代大鼠神经细胞培养F-肌动蛋白泪点的定量。 标毒肽对大型和小型丝（F-肌动蛋白）相似的亲和力，但不绑定到单体球状肌动蛋白（G-actin的），不像有些肌动蛋白抗体，1。鬼笔环肽非特异性结合是微不足道的，因此，细胞成像过程中提供最小的背景。鬼笔环肽是比通常被用于标记细胞蛋白为荧光显微镜，其允许由鬼笔环肽F-肌动蛋白的更强烈标记抗体小得多。因此，在神经元F-肌动蛋白本地化的详细图像可以通过使用标记的鬼笔环肽而获得的。 毒伞素（F-肌动蛋白），神经元树突染色产生离散的"热点"或明亮的"泪点"，它代表了各种树状结构，包括成熟的刺，无刺的突触2和不成熟的刺。未成熟的棘包括薄丝状伪足和某些形式的贴剂的形态，并且可以代表spinogenesis 3的启动。未成熟的刺和非多刺补丁缺乏PSD95 4。从而在生产F-肌动蛋白导致不仅刺后续变化，而 ​​且还附加树枝状结构的变化，使得鬼笔环肽进行调查synaptodendritic完整性5-7的重要工具。一般情况下，鬼笔环肽阳性（F-肌动蛋白）泪点的数字反映了积极的突触之间的平衡（兴奋和抑制），肌动蛋白和突触的稳定性8。 虽然研究特异性T是非常重要的突触（即，兴奋性刺）的ypes，当治疗的靶标是未知有必要首先估计各种树枝状结构的总体完整性。由于F-肌动蛋白是树突棘和其它结构，包括抑制性突触，改变的数目F-肌动蛋白泪点的可指示synaptopathy的主要组成部分。这synaptopathy然后可用于更具体的变化进一步调查。我们的检测多种类型的突触/结构的量化方法产生的树突突触的改变（增加和减少）以下的各种实验性治疗的总体估计。

<table><tbody><tr><td>35 mm Glass Bottom Dishes No. 1.5 coverglass</td><td>MatTek Corporation</td><td>P35G-1.5-20-C</td><td></td></tr><tr><td>DMEM/F12 medium</td><td>Life Technologies</td><td>10565-018</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P9155</td><td></td></tr><tr><td>Boric acid</td><td>Sigma</td><td>B0252</td><td></td></tr><tr><td>Borax</td><td>Sigma</td><td>B9876</td><td></td></tr><tr><td>GlutaMax</td><td>Life Technologies</td><td>35050-061</td><td>100X</td></tr><tr><td>Glucose</td><td>VWR</td><td>101174Y</td><td></td></tr><tr><td>HBSS</td><td>Sigma</td><td>H4641</td><td>10X</td></tr><tr><td>Neurobasal medium</td><td>Life Technologies</td><td>21103-049</td><td></td></tr><tr><td>B-27 supplement</td><td>Life Technologies</td><td>17504-044</td><td>50X</td></tr><tr><td>Antibiotic-Antimycotic solution</td><td>Cellgro</td><td>30004CI</td><td>100X</td></tr><tr><td>Sodium Bicarbonate</td><td>Life Technologies</td><td>25080</td><td></td></tr><tr><td>Vannas Scissors</td><td>World Precision Instruments</td><td>500086</td><td></td></tr><tr><td>Iris Scissors</td><td>World Precision Instruments</td><td>500216</td><td></td></tr><tr><td>Iris Forceps</td><td>World Precision Instruments</td><td>15914</td><td></td></tr><tr><td>Dumont #7 Forceps</td><td>World Precision Instruments</td><td>14097</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>World Precision Instruments</td><td>14095</td><td></td></tr><tr><td>ProLong Gold</td><td>Life Technologies</td><td>P36930</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Cover glass</td><td>VWR</td><td>631-0137</td><td></td></tr><tr><td>AlexaFluor 488 Phalloidin</td><td>Life Technologies</td><td>A12379</td><td></td></tr><tr><td>Normal horse serum</td><td>Life Technologies</td><td>26050-070</td><td></td></tr><tr><td>Chicken polyclonal anti-MAP2</td><td>abcam</td><td>Ab92434</td><td></td></tr><tr><td>Alexa Red 594-conjugated goat anti-chicken IgG</td><td>Life Technologies</td><td>A11042</td><td></td></tr><tr><td>NucBlue Live cell stain ReadyProbes Reagent Hoescht 33342</td><td>Life Technologies</td><td>R37605</td><td></td></tr><tr><td>NIS-Elements software package</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE2000-E inverted fluorescent computer-controlled microscope&amp;nbsp;</td><td>Nikon Instruments</td><td></td><td></td></tr><tr><td>0.2 &amp;mu;m Nalgene nylon membrane filter</td><td>Fishersci</td><td>151-4020</td><td></td></tr></tbody></table>

quantification of filamentous actin (f-actin) puncta in rat cortical neurons

肌动蛋白丝状蛋白（F-肌动蛋白）起着spinogenesis，突触可塑性，突触稳定的重大作用。树突状F-肌动蛋白丰富的结构变化表明突触的完整性和连通性的改变。在这里，我们为主要培养大鼠皮层神经元，鬼笔环肽染色F-肌动蛋白泪点，和随后的量化技术的详细协议。首先，E18大鼠胚胎的额叶皮层解离成低密度细胞培养，然后在体外生长至少12-14天的神经元。以下实验性治疗，皮质神经元染色AlexaFluor 488鬼笔环肽（标记树枝状F-肌动蛋白泪点）和微管相关蛋白2（MAP2;验证神经元细胞和树突完整性）。最后，专门的软件，用于分析和量化随机选择的神经元树突。 F-肌动蛋白丰富的结构被确定二阶树突分支（长度范围25-75＆＃181;米），连续MAP2免疫。这里介绍的协议将是调查到实验处理后续树突突触结构的变化的有用方法。

在本发明的方法，我们首先培养的大鼠密度低35毫米的玻璃底菜，这使我们能够识别单个神经元的树突皮层神经元。在图1中，微分干涉对比（DIC）图像显示在天4，6，10，14， 在体外 21和27显影胎大鼠皮质神经元的形态学变化。注意，长度和树突的数量与培养的大鼠原代神经元的成熟增加。神经元在实验中只后14天成熟使用。 <p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons at JoVE.com

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

大鼠皮层神经元丝状肌动蛋白（F-肌动蛋白）泪点的量化

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

quantification-filamentous-actin-f-actin-puncta-rat-cortical

Research

JoVE Journal

Neuroscience

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

12.0K 次观看.  University of South Carolina. Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

视频: Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

突触量化:基于一个免疫细胞化学检测，以量化突触数量

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

科研

神经科学

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

罗丹明-肌动蛋白标记F -肌动蛋白在通透的神经生长锥刺完

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.

DetectSyn:一种快速、无偏倚的荧光方法，用于检测突触密度的变化

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is ...

生物化学

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

封闭系统中脑切片长期培养中精确定位和重复间歇成像的改进辊筒法

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

用于评估啮齿动物和人类脑组织中行为素聚合状态的基于时间的荧光光谱分析

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy (3D-SIM)

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to the maintenance of neuronal polarity by sorting somato-dendritic cargo. A membrane periodic skeleton (MPS) comprising periodic actin rings provides a scaffold for anchoring various AIS proteins, including structural proteins and different ion channels. Although recent proteomic approaches have identified a considerable number of novel AIS components, details of the structure of the MPS and the roles of its individual components are lacking. The distance between individual actin rings in the MPS (~190 nm) necessitates the employment of super-resolution microscopy techniques to resolve the structural details of the MPS. This protocol describes a method for using cultured rat hippocampal neurons to examine the precise localization of an AIS protein in the MPS relative to sub-membranous actin rings using 3D-structured illumination microscopy (3D-SIM). In addition, an analytical approach to quantitively assess the periodicity of individual components and their position relative to actin rings is also described.

Measuring Properties of the Membrane Periodic Skeleton of the Axon Initial Segment using 3D-Structured Illumination Microscopy 3D-SIM

The present protocol describes a method to visualize and measure actin rings and other components of the membrane periodic skeleton of the axon initial segment using cultured rat hippocampal neurons and 3D-structured illumination microscopy (3D-SIM).

使用3D结构照明显微镜（3D-SIM）测量轴突初始段的膜周期性骨架的性质

The axon initial segment (AIS) is the site at which action potentials initiate and constitutes a transport filter and diffusion barrier that contribute to ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

软件辅助跟踪动态细胞突变中蛋白质浓度的图形用户界面

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

生物学

Quantification of Filamentous Actin Puncta in Rat Cortical Neurons

Source: Li, H., et al. <a href="https://app.jove.com/v/53697">Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.</a> J. Vis. Exp. (2016)
This video demonstrates the procedure for fluorescently staining rat cortical neurons for microscopy. It targets F-actin and dendritic proteins to analyze structural integrity and calculate F-actin density, which is crucial for studying neuronal function and health.

大鼠皮质神经元中丝状肌动蛋白点的定量

1. Fluorescent Labeling and Immunocytochemistry
Note: The immunofluorescent labeling of primary cortical cell cultures was carried out in glass bottom 35 ...