我们非常感谢我们的的合作者克努特Holthoff，亚瑟Konnerth和Marco Canepari参加这种技术处于最初的发展以及对Leslie M.勒夫好心提供染料。由国家科学基金会资助IOS-0817969，国立卫生研究院资助NS068407和M136043和在耶鲁大学Kavli研究所神经科学支持。

1。设备安装步骤1.1。影像设定记录电压敏感染料信号的关键是适当的安装设计。我们使用配备三个摄像头的直立显微镜（BX51WI奥林巴斯或蔡司AxioExaminer的）。是专为照明单个神经元在大脑切片在落射荧光，激发光的宽视场显微镜模式，可以使用尼康60X/1.0 NA NA或蔡司63X/1.0的水浸渍目标的设置。我们的显微镜是用螺栓固定到一个隔振表，并配备与机动项目可动阶段。每个显微镜配备了3个摄像头端口。 One相机端口有一个标准的高空间分辨率的CCD摄像头，用于红外DIC的视频显微镜（IR-1000，美国），打格MTI。第二个摄像头端口有一个快速的摄像头数据采集（高达20 kHz的帧速率）与相对较低的空间分辨率（80×80像素），但具有出色的动态范围（14位）和极低的读噪声（NeuroCCD-SM，RedShirtImaging有限责任公司，迪凯特，GA）。第三相机端口具有高空间分辨率的CCD照相机（PixelFly，1392x1024像素; PCO AG，德国）上安装​​的纺丝盘的共焦扫描仪（CSU-10，日本横河，）用于收集的共焦图象的z栈详细的形态学重建染的细胞。一个倍频二极管泵浦Nd：YVO4连续波激光（400 mW）的发射在532 nm电厂（MLL-III/400兆瓦; CNI，长春，中国）是的源激发光。的2毫米直径的由快门（LS6;文森Associates）的选通的激光束被引导到导光耦合到一个单端口设计背面孔径装得过满的落射荧光冷凝器（TILL Photonics公司，Gräfelfing，德国）通过显微镜目标和提供接近的物体面的均匀的照明。激光被用于代替常规的氙弧灯的V 间成像的灵敏度最大化：（1）使用一个单色的前引用光的吸收光谱的红翼V M灵敏度的染料9，10和（2）增加的激发光的强度以外的水平，可以通过以下来实现的电弧灯最大化。由与中心波长为560nm和记录的荧光通过610nm的屏障过滤器（肖特RG610）通过的分色镜的激发光被反射到该制剂。光强度的增加和使用接近最佳的单色激发波长的综合效果，在电压成像的灵敏度相比，以前的测量6 -约50的一个因素是一个显着的改善。 步骤1.2。调整为均匀的照明使用的荧光滑标准（绿色激发/红色发射）。插入适当的中性密度过滤器，以便在激光束光路的不饱和的CCD。聚焦目标的表面上在幻灯片上。调整石英导光体，在前面的激光发射器的目标位置的接收端，和调整的输出端连接到显微镜使用适当的致动器上的落射荧光冷凝器来实现的导光的位置为中心的和均匀的视场照明。 步骤1.3。确定振动噪声将一个小的黑色墨水标记的表面上的荧光幻灯片。记录光强度与NeuroCCD在连续记录模式。在黑暗边缘的黑色墨迹，聚焦目标。记录光的强度在2 kHz的帧速率约100毫秒。显示分数的光强度的痕迹（ΔF/ F）从接收的光均匀地照射的区域，从〜20沿墨水标记的边缘的像素〜20像素的空间平均值。在录音像素高对比度的边缘多余的噪音反映了振动噪声系统。 步骤1.4。隔振它是强制性的，通过使用振动隔离表媲美实验记录条件的光强度的散粒噪声的电平以下时，以减少振动。调整的振动隔离表，直到从覆盖的图像的锐利的边缘的像素的光强度的振动噪声可以忽略不计的。没有运动部件（机械快门，风扇）的设备可被安装在桌子上。从设备连接到其他组件不振动隔离在桌子上的电缆必须是宽松的，使他们不传输机械振动的显微镜。 2。选择一个适当的神经元为V 米影像步骤2.1。神经元的选择根据标准程序，使脑切片。使用鼠标线，我的神经细胞表达EGFPnterest。一个旋转盘共聚焦系统，可视化EGFP标记的切片中的神经元。选择为V 米成像神经元与完整的树突状/轴索树木，并与正在运行的进程的切片的表面平行且靠近。这不能被实现，在野生型小鼠，因为轴突和薄的枝晶，在大多数情况下，是不可见的在DIC的显微镜模式。 3。将神经元与电压敏感染料步骤3.1。填写的补丁吸管填充的前端与无染料的细胞内溶液的玻璃的补丁移液器从通过施加负压力约15秒的电极锥度的三分之二。在管尖中的染料的免费的解决方案是必要的，以防止泄漏的染料到增加的背景荧光，并极大地减少了信号噪声比的切片。返回填充含有的膜不能渗透电压敏感染料JPW3028溶解的溶液与所述电极在细胞内的溶液（0.8毫摩尔）。 JPW3028，最成功的电压探头的细胞内的应用程序，是双带正电的类似物ANEP一系列的亲脂性的苯乙烯基染料，仍然是足够的水溶性要用于显微注射。二-乙基类似物，该染料具有几乎相同的特性（包括电压灵敏度）和市售JPW1114（ 见表1）。我们准备20毫米原液在蒸馏水中H 2 O。 50μl的等分试样的原液冷冻保存于-20℃下对于最终的染料浓度为0.8 mM的，2微升原液溶解在50μl的细胞内溶液在实验当天。的库存中的染料溶液是稳定的，可以在室温下保持数个月。因此，我们在室温下保持50微升等分，直到它被用完。 步骤3.2。迅速建立一个千兆密封<p claSS =“jove_content”&gt;修补程序先前选定的神经元，并允许自由扩散的染料从体膜片微电极胞体在整个单元配置为20-45分钟。它是必不可少的，以获得尽可能快的密封，因此，在管尖中的染料溶液的量可以很小。开始实行快速修补而不在电极中的染料。接着，实践的修补和装载神经元与染料不污染周围的组织的情况下，使用最小量的无染料溶液中的前端的与目标。与实践，它是可以在1-2分钟之内建立一个密封。 步骤3.3。监视水平的染色在整个细胞结构的染料扩散的过程中，在电流钳模式下记录诱发电位监测的神经元的生理状态。此外，监测通过记录的2 k的帧速率从胞体的静息的光强度（RLI）染色的量<html> Hz和在全光强度调节中性密度过滤器（从400 mW的激光，我们使用的激光的光强度的0.04％）的一小部分。继续染料的加载过程，直​​到行动的潜力开始扩大，通常在20-40分钟，取决于电极的大小和阻力。 尖峰的扩大是完全可逆的，最有可能是由于容性负载体膜中的染料的饱和浓度的影响。尖峰的波形的完全恢复后的染料浓度为整个神经元平衡。 步骤3.4。删除的染料电极在染色期间结束，修补程序移液管小心地拉出距离苏摩在确保从全细胞过渡外满分补丁配置的过程中达到的电压钳位配置。 步骤3.5。等待染料扩散耳鼻喉科“&gt;的一个额外的1.5-2小时，在室温下孵育切片，以允许的电压敏感染料扩散到神经元的过程。后显着量的染料扩散远离苏摩成树突和轴突的过程，的波形的AP完全恢复。 4。光学记录步骤4.1。选择一个与细胞成像找到染色的神经元胞体在低光照水平荧光和重新修补贴片电极下DIC的标准（染料）的神经元。可视化神经元的过程，低光照下在10-40赫兹电压成像在CCD的连续记录模式中的帧速率的水平。减少光的中性密度滤镜感兴趣的对象，可视化所需的最低水平。染色的神经元的定位过程中，我们使用0.01％的400 mW的激光强度。使用XY阶段，定位在t神经元过程中的利益他中的摄像区域。保护苏摩从激励光用的局部封闭的显微镜的视场光阑光圈。屏蔽胞体由高强度激发光在录制过程中会显着减少光动力损伤。 步骤4.2记录膜电位瞬态相关的光信号记录光信号backpropagated个人树突分支的AP。记录光信号，在轴突向受影响。光学记录到在树突棘backpropagating接入点的信号。使用适当的准确重建信号波形的帧速率，并保持尽可能短的记录期间，暴露于高强度的激励光，以尽量减少染料漂白和光动力损伤。例如，检查序列的萌生和扩展的单个动作电位在轴突需要记录周期为5-10毫秒。激发光强度在记录过程中的性是一方面上的信号 - 噪声比，和染料的褪色程度和另一方面的光动力损伤之间的一种折衷。我们使用100％的强度为400 mW的激光在录音时的照明面积的直径为300微米的神经轴突的长款。当激发光被聚焦到一个的树突棘成像30微米直径的区域，我们使用的激光的光强度的10-25％。所需的时间记录的最佳激发光强度是一个重要的决定因素;较短的记录时间允许更高的光强度。最好凭经验确定最佳的光照强度为每个制备和测量设置。 5。数据分析步骤5.1。已知的错误，更正原始数据使用NeuroPlex的程序（RedShirtImaging）编写的IDL（ITT视觉信息Solut进行分析和显示数据离子，博尔德，科罗拉多州）和自定义Visual Basic例程。低光水平的条件下，背景荧光的ΔF/ F信号的大小成为一个显着的行列式。此效果，首先被纠正的原始数据减去从一个未染色的区域中的片上确定的平均背景荧光强度。随后，信号校准软件，用于纠正在AP启动的时间抖动以及可能的小运动的准备期间平均。在时域，AP信号对齐的电记录的AP在每个试验，以在开始的平均获取的参考信号（ 图1B）通过交叉相关。在空间域中，图像对准在两个维度脱机的图像交叉相关的，以补偿可能的小的横向运动的准备。正确的聚焦在z维度上的图像之前，验证每个单独的试验; SMaL公司<html>升调整往往是必要的。空间和时间上对齐的信号的平均值，如在图1B中所示。由于染料漂白，缓慢变化的光强度除以来自试验用无刺激（ 图1B）的记录由一个适当的双指数函数的数据进行校正。从一组数据点，用三次样条插值，分段连续曲线通过每个数据点的AP信号的波形重建。从胞体中，电的AP信号进行比较，以确认电压敏感染料信号跟踪膜电位没有显着的失真在毫秒时间尺度的光的AP信号为如图3B中所示，从相邻的轴突的小丘。这两个信号叠加关系十分密切，使散粒噪声光学记录。 les/ftp_upload/4261/4261fig1.jpg“/&gt; 图1。数据分析（动画）。 （A）上图：高分辨率的共聚焦图像的彩色神经元轴突在记录位置。记录电极附着胞体和细胞外刺激电极基枝晶示意性地示出。外，突触刺激诱发的动作电位。下图：低空间分辨率的荧光图像的轴突用于V M成像。通过CCD（B）的电极记录从SOMA（黑色痕迹），光学录音AIS（红色痕迹），并从郎飞结点的（绿色的痕迹） 。顶部的痕迹：原始数据来自9个试验，在AP启动的时间抖动。第二排的痕迹：时间对准信号。第三排：平均信号的痕迹。第四排的痕迹：漂白校正。底部的痕迹：三次样条插值的时间平滑的一次。

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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+multisite+optical+measurement+of+membrane+potential.">Fast multisite optical measurement of membrane potential.</a> A practical guide to technology for quantitative real-time analysis. , 389-404 (1993).</li><li>Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dendritic Spines. , (2010).</li></ol>

德扬Zecevic宣称他是共同拥有的RedShirtImaging有限责任公司，该公司专注于高转速，低噪音CCD相机采用电压敏感染料记录。所有其他的作者报告说目前的研究没有经济利益或潜在的利益冲突。

本文介绍了一种电压敏感染料的记录方法监测与亚微米和亚毫秒级的时空分辨率的单个神经元的电活动。激光激发在接近最优波长（关于信号的大小）提高了灵敏度的记录〜50比以前的方法的一个因素。目前的灵敏度，使电信号监测单个神经元的所有部分，包括树突，轴突，轴突抵押品和轴突终端以及个人的树突棘。目前的灵敏度，可以进行录音膜电位瞬态高达20 kHz的帧速率。适度的信号平均（4-...

<table border="1"><tbody><tr><td> 的组件的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td></td><td></td><td></td><td> 安装组件 </td></tr><tr><td>直立显微镜</td><td>奥林巴斯公司</td><td> BX51WI </td><td>有三个摄像头的端口</td></tr><tr><td>电动移动式舞台</td><td>锡斯基尤</td><td> MXOPi.2 </td><td></td></tr><tr><td>落射荧光冷凝器奥林巴斯BX51 </td><td> TILL光电</td><td> 0000-560-11659 </td><td></td></tr><tr><td>直立显微镜</td><td>卡尔·蔡司，LLC </td><td> AxioExaminer D1 </td><td>有三个摄像头的端口</td></tr><tr><td>电动顶板</td><td> Scientifica有限公司</td><td> MMBP </td><td></td> &lt;/ TR&gt; <tr><td>落射荧光冷凝器蔡司AxioExaminer </td><td> TILL光电</td><td></td><td></td></tr><tr><td>摄像头数据采集</td><td> RedShirtImaging有限责任公司</td><td> NeuroCCD-SM </td><td>高速，低读出噪声</td></tr><tr><td> CCD IR-DIC </td><td> DAGE-MTI </td><td> IR-1000 </td><td></td></tr><tr><td>旋转盘共焦扫描仪</td><td>横河</td><td> CSU-10 </td><td></td></tr><tr><td>共焦扫描仪的高空间分辨率CCD </td><td> PCO AG </td><td> PixelFly </td><td> 1392x1024像素</td></tr><tr><td> DPSS CW激光（532nm） </td><td>北方光电技术。有限公司</td><td> MLL-III-532为400mW </td><td>激发光源</td></tr><tr><td>多模光纤发射器</td><td>锡斯基尤</td><td> SM-CFT </td><td></td></tr><tr><td>光导</td><td> TILL照片网卡</td><td> 0000-515-11524 </td><td></td></tr><tr><td>快门</td><td>文森特联营公司</td><td> LS6 </td><td></td></tr><tr><td>隔振表</td><td>减k科技</td><td> MK26 </td><td></td></tr><tr><td></td><td></td><td></td><td> 具体试剂 </td></tr><tr><td>二-2-ANEPEQ（JPW 1114） </td><td> Life Technologies公司</td><td> D-6923 </td><td>电压敏感染料</td></tr><tr><td> Crym-EGFP鼠标线</td><td> GENSAT（MMRRC） </td><td>联合TG（Crym-EGFP）GF82Gsat/Mmcd </td><td>在第5层皮层神经元稀疏表达EGFP </td></tr></tbody></table>

voltage-sensitive dye recording from axons, dendrites and dendritic spines of individual neurons in brain slices

了解单个神经元的生物物理特性和功能组织，以及他们是如何处理信息的理解大脑是如何工作的根本。任何神经细胞的主要功能是处理电信号，通常是从多个源。电性能的神经元的过程是极其复杂的，动态的，并且，在一般情况下，不可能预测在没有详细的测量。为了得到这样的测量1，理想的情况下，能够监控，在多个地点，亚阈值事件，因为它们从原产地的网站上神经元的过程和summate在特定的位置，影响动作电位开始。在任何神经元由于采用电极测量技术的限制，这一目标一直没有实现。为了克服这个缺点，它是非常可取的成像技术，允许广泛的平行recordin补充补丁电极的方法GS来自全国各地的神经元。在这里，我们描述了这样的技术-光记录的有机电压敏感染料（Ⅴ 米成像） -其特征在于由亚毫秒和亚微米分辨率的膜电位的瞬态。我们的方法是基于对电压敏感的分子探针2的开拓性工作。的初始技术的许多方面已不断提高超过几十年3，5，11。此外，以前的工作记录的V 米成像的两个基本特征。首先，荧光信号是线性正比于在整个生理范围（-100 mV到+100 mV，10，14，16）的膜电位。其次，装载与这里所使用的电压敏感染料（JPW 3028）神经元没有检测到的药理作用。记录扩大在穗染料吸附量是完全可逆的，7。此外，实验证据表明，它是能够得到一个显著的数量多达数百录音之前，任何检测到的光毒性作用4，6，12，13。目前，我们利用的高超近最佳波长的灵敏度最大化的V 米成像技术的激光光源的亮度和稳定。目前的灵敏度，允许多个站点的光学录音的V M瞬变的一个神经元的所有部分，包括轴突和轴突抵押品，终端树突分支，以及个人的树突棘。所获取的信息的信号相互作用的可定量分析以及直接可视化在一部电影的形式。

激光共聚焦显微镜成功应该能够清楚识别的完整的神经元突起的切片的表面，这是在一个平面上的重点。这是适当的电压成像之前的电压敏感染料荷载的神经细胞的选择是至关重要的。皮质切片表达EGFP（Crym转基因小鼠品系）的L5锥体神经元的共焦图象的一个例子是在图2中所示。单个神经元的轴突都清晰可见。细胞在一个平面上的焦点的切片的表面接近与的长期完好轴突（白箭头）被...

Watch this Scientific Journal Video about Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices at JoVE.com

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

一种成像技术用于监测具有亚微米的空间和亚毫秒时间分辨率的膜电位的变化进行说明。技术，基于电压敏感染料激光激发，使测量信号的轴突和轴突抵押品，终端树突分支，和个人的树突棘。

电压敏感染料记录单个神经元在大脑切片的轴突，树突和树突棘

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

voltage-sensitive-dye-recording-from-axons-dendrites-dendritic-spines

Research

JoVE Journal

Neuroscience

16.6K 次观看.  Yale University School of Medicine. 一种成像技术用于监测具有亚微米的空间和亚毫秒时间分辨率的膜电位的变化进行说明。技术，基于电压敏感染料激光激发，使测量信号的轴突和轴突抵押品，终端树突分支，和个人的树突棘。

视频: Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

从啮齿动物的神经元和非人类灵长类动物的脑片DiOLISTIC标签

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

科研

神经科学

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

蟹电压敏感染料Stomatogastric神经节神经元的光学成像

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1 . We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B and C). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3 .The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6, the frequency of which is modulated by visual cues from conspecifics2 and the neuropeptide kisspeptin 15. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3 linked to enhanced green fluorescent protein7 to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6.

In this video, we will demonstrate how to record electrical activity from identified single neurons in a whole brain preparation, which preserves complex neural circuits. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with a fluorescent protein for identification in the intact brain preparation.

鉴定转基因鱼的完整的大脑中的神经元的电活动记录

Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

成像大鼠原代海马神经元树突棘使用结构照明显微镜

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

七鳃鳗网状脊髓轴突急性分解到从单个功能突触前终端剥离面膜启用记录

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ fluctuations can occur through a variety of membrane and intracellular mechanisms and play a crucial role in the induction of synaptic plasticity and regulation of dendritic excitability. Hence, the ability to record different types of Ca2+ signals in dendritic branches is valuable for groups studying how dendrites integrate information. The advent of two-photon microscopy has made such studies significantly easier by solving the problems inherent to imaging in live tissue, such as light scattering and photodamage. Moreover, through combination of conventional electrophysiological techniques with two-photon Ca2+ imaging, it is possible to investigate local Ca2+ fluctuations in neuronal dendrites in parallel with recordings of synaptic activity in soma. Here, we describe how to use this method to study the dynamics of local Ca2+ transients (CaTs) in dendrites of GABAergic inhibitory interneurons. The method can be also applied to studying dendritic Ca2+ signaling in different neuronal types in acute brain slices.

We present a method combining whole-cell patch-clamp recordings and two-photon imaging to record Ca2+ transients in neuronal dendrites in acute brain slices.