Name: imaging neurons within thick brain sections using the golgi-cox method
Uploaded: 2017-04-18T13:00:00.000+00:00
Duration: 10 min 26 s
Description: Watch this Scientific Journal Video about Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method at JoVE.com

这项工作是由一个发现格兰特CDCB从自然科学和加拿大（NSERC）工程研究理事会，加拿大创新基金会（CFI项目编号30381），约翰·R·埃文斯领导基金研究基础设施补助金CDCB，并支持发现格兰特NJM从NSERC。 ELL是由安大略研究生奖学金和安大略兽医学院的奖学金OVC圭尔夫大学的支持。 CDS是由NSERC本科生助研支持。 ALM是由NSERC亚历山大·格雷厄姆·贝尔奖学金和安大略兽医学院在圭尔夫大学的奖学金OVC支持。

成年雌性CD1系小鼠在本研究中使用。类似的染色可以在不同的年龄段使用两种性别来完成。实验动物照顾根据原则和加拿大动物保护协会的指导方针，以及实验方案经圭尔夫大学动物保护委员会的大学。 1.高尔基考克斯染色<ol><li> 脑的高尔基-考克斯浸渍 <ol><li>由重铬酸钾和铬酸钾成高品质的水分别溶解使1％的高尔基体考克斯溶液（W / V）的重铬酸钾，0.8％（W / V）的铬酸钾和1％（W / V）氯化汞。混合溶液中，添加氯化汞和过滤器使用1级滤纸最终溶液。存储长达一个月在黑暗中的解决方案。 </li><li>麻醉，用5％异氟醚的鼠标。 </li><li>断头安乐死鼠标，迅速取出大脑。 </li><li>放置大脑到含有17毫升的高尔基考克斯溶液20mL玻璃闪烁瓶中，并在室温在黑暗中孵育25天。 </li><li>通过在4℃下将其置于含有40毫升蔗糖的冷冻保护剂的50mL锥形管中（30％（W / V）蔗糖的0.1M磷酸盐缓冲液，pH 7.4）在黑暗中24小时Cryoprotect大脑。 注:以下可选三个步骤可被用作替代的主要协议，为了冻结所述的脑在此阶段对于长期储存。 </li><li>通过将其浸入已预冷在干冰上200毫升异戊烷冻结整个大脑。 </li><li>放置冷冻大脑进入在黑暗中的50mL锥形管中，并储存在-80℃。 </li><li>当准备好继续时，通过将其放置到含有40毫升在黑暗中蔗糖的冷冻保护剂在4℃下24小时的50mL锥形管解冻大脑。 </li></ol></li><li> 脑切片 <ol><li>从蔗糖和集团取出大脑ķ它用于通过切断使用刀片小脑和在脑的剩余尾端留下一平边缘切片。 </li><li>热琼脂（3％（重量/体积）的水），直到它被熔化并放冷，直到它是稍微高于其熔点。 </li><li>放置在脑以其尾端面朝下一个小一次性称重盘中，并添加熔融的琼脂足够量的以覆盖大脑。 </li><li>一旦琼脂凝固后，修剪多余琼脂留下大约2 - 4毫米大脑周围和胶水大脑使用乙基氰基丙烯酸酯胶少量其尾端面朝下一个振动切片机的阶段。 </li><li>填充蔗糖的冷冻保护剂的足量的振动切片机的阶段区以覆盖所述脑，和部分中的脑在400层厚 - 使用86赫兹的振动频率为500μm（取决于大脑区域待检查）和为0.125mm / s的刀片前进速度。 </li><li>使用小油漆刷，地点脑切片成含有市售的网状底部插入和6孔组织培养板的孔中的预填充用10mL的6％的0.1M磷酸盐缓冲液，pH 7.4（W / V）蔗糖。 </li><li>在4℃CO / N孵育在黑暗部分。 </li></ol></li><li> 开发脑切片 <ol><li>使用网状底部插入，在0.1M磷酸盐缓冲液，pH 7.4中转移的脑切片到一个新的孔含有5毫升的2％（W ​​/ V）多聚甲醛（PBD）。孵育上的摇杆以低速在黑暗中在RT移动15分钟。 </li><li>通过将它们转移到含有5毫升水新井洗部分两次。洗上的摇杆以中等速度在黑暗中在RT移动5分钟。 </li><li>转移部分到含有5毫升的2.7％（V / V）氢氧化铵一个新井。孵育上的摇杆以缓慢的速度在黑暗中在RT移动15分钟。 </li><li>通过将它们转移到含有5毫升水新井洗部分两次。 Ø洗净呐摇杆以中等速度在黑暗中在RT移动5分钟。 </li><li>转印部到一个新的孔含有5毫升固定剂A的（见材料表 ），其具有在水中的10倍稀释从其原始购买浓度。孵育上的摇杆以缓慢的速度在黑暗中在RT移动25分钟。 </li><li>通过将它们转移到含有5毫升水新井洗部分两次。洗上的摇杆以中等速度在黑暗中在RT移动5分钟。 </li></ol></li><li> 安装脑切片 <ol><li>使用小油漆刷，安装部到显微镜载玻片上。除去过量的水和琼脂使用镊子和小组织。确保所有琼脂和脱水继续之前除去。 </li><li>允许部分空气干燥在室温约45分钟（400个微米的部分）或90分钟（500个微米的部分）。 注:干的时间和适当的水平是关键的，并且可能需要在每一个都被确定实验室取决于环境温度和湿度水平。过短的干燥时间，导致在随后的脱水步骤脱落幻灯片的部分，和太长的干燥时间导致部分开裂。部分将仍然显得有光泽，在干燥的适当水平。 </li><li>通过将滑动到科普林染色瓶当指示用甲酚紫染色切片。 注:此可选步骤可以采用一种从未冰冻切片，以染色用甲酚紫神经元细胞核。我们发现，清理和甲酚紫孵育前补水部分导致甚至染色和跨节低背景。 <ol><li>在清除剂5分钟放置。重复一次。 </li><li>在100％乙醇放置5分钟。重复一次。 </li><li>在95％乙醇中放置水，再用75％乙醇在水中，然后在水中50％的乙醇，每次2分钟。 </li><li>在水中放置5分钟。 </li><li>在0.5％的地方（W / V）CRESYL在水中呈紫色7分钟。 </li><li>在水中放置2分钟。重复一次。 </li></ol></li><li>通过将滑动到科普林染色罐子所示脱水部分。 <ol><li>在50％乙醇中放置水，再用75％乙醇在水中，然后在水中95％的乙醇，每次2分钟。 </li><li>在100％乙醇放置5分钟。重复一次。 </li><li>在清除剂5分钟放置。重复一次。 注意:这些脱水和清洁时间足以在这个手稿中描述处理小鼠的大脑。然而，我们观察到对其他物种，包括大鼠和牛鹂属，最终结算步骤可能需要被延长至15分钟总。 </li></ol></li><li>使用无水安装平台盖玻片部分。 </li><li>允许滑动以水平地在黑暗中在室温下至少5天干燥。 </li></ol></li></ol> 2.厚脑切片内成像彩色神经元<ol><li> 捕获图像堆栈 </STRONG&gt; <ol><li>打开显微镜灯泡，相机和阶段控制器。 </li><li>打开显微镜软件（ 例如 ，Neurolucida）。 </li><li>放置在显微镜载物台上的滑动。 </li><li>使用低放大倍数的物镜如1.25X 0.4 NA PlanAPO或4X 0.16 NA捕获脑切片的2D宽视图图像<ol><li>焦点图像和调整相机设置包括曝光时间和白平衡。 </li><li>上一节创建左键点击一个参考点的任何位置</li><li>通过选择图像采集窗口内"获取单个图像"捕获图像。 </li></ol></li><li>捕获包含感兴趣的神经元（多个）区域的中间分辨率图像栈，使用10X 0.3 NA UPlan FLÑ目标。 <ol><li>聚焦图像和调整相机设置包括曝光和白平衡。 </li><li>通过集中到一个部分的顶部设置用于图像堆栈中的上和下边界第二选择"设置"图像采集窗口内旁边的"堆栈的顶部"，然后聚焦到段的底部，并选择所述图像采集窗口内"设置"旁边的"栈的底部"。 </li><li>由图像采集窗口内输入"5微米"旁边的"距离图像之间的"设定为5μm的步距。 </li><li>由图像采集窗口内选择"获取图像堆栈"捕获图像栈。 </li><li>重复上述步骤，以捕获感兴趣的整个区域，确保所有图像栈至少10％，在X和Y轴重叠。 </li></ol></li><li>捕获包含感兴趣的神经元区域的高分辨率图像栈，使用30X 1.05 NA硅油浸泡的目的。 <ol><li>申请3 - 硅浸油4滴于载玻片并把目标在幻灯片，确保能够使目标与爱之间的接触湖</li><li>聚焦图像和调整相机设置包括曝光和白平衡。 </li><li>通过集中到该部分的顶部，并选择"设定"设定为图像堆栈中的上和下边界旁图像采集窗口内，然后"堆的顶部"聚焦到段的底部，并选择"设置"图像采集窗口内旁边的"栈的底部"。 </li><li>由图像采集窗口内输入"1微米"旁边的"距离图像之间的"设置为1微米的步距。 </li><li>由图像采集窗口内选择"获取图像堆栈"捕获图像栈。 </li><li>重复上述步骤，以捕获感兴趣的整个区域，确保所有图像栈至少10％，在X和Y轴重叠。 </li></ol></li><li>保存数据文件和TIFF格式保存所有图像文件进行外部处理。 </li></ol> &lt;/ LI&gt; <li> 创建Z-投影图像和图像的剪辑画面 <ol><li>创建ImageJ的Z-投影图像<ol><li>已经安装了生物格式插件打开ImageJ软件。 </li><li>选择"插件" - &gt;"生物格式" - &gt;"生物格式进口商"。 </li><li>选择图像栈要打开的文件。 </li><li> &gt;"类型" - - &gt;"RGB颜色"一旦文件被打开，通过选择"图像"更改格式为RGB。 </li><li>通过选择 "图像" 创建Z-投影 - &gt; "堆栈" - &gt; "Z项目..."。 </li><li>保存的二维Z-投影图像为TIFF文件。 </li></ol></li><li>创建感兴趣整个区域的二维图像蒙太奇。 <ol><li>打开软件（ 如 Adobe公司的Photoshop）。 </li><li>选择 "文件" - &gt; "自动完成" - &gt; "的Photomerge"。 </li><li>选择"浏览"，然后添加所有IMAGES文件合并。 </li><li>确保"混合图像一起"被选中，然后选择"OK"。 </li><li>保存的利益为TIFF文件的整个区域的产生蒙太奇图像。 </li></ol></li></ol></li></ol>

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作者宣称，他们没有竞争的经济利益。

我们在这里介绍一个高尔基考克斯染色协议了两个有用的变种沿着厚的脑切片中神经元的可视化。正如代表结果所示，使用具有长800μm的工作距离的高分辨率目标的允许整个神经元在整个500μm的切割脑切片的深度可靠可视化。比较厚的脑切片的这项研究增加了任何类型的神经元染色完全包含切片，这是长而复杂的顶端树突树锥体神经元是特别重要内的概率。例如，在啮齿动物的脑中的冠状切片，?...

单个神经元的组织样本中的可视化允许的神经元形态特征的原位分析，已显著提高了我们的大脑的理解，以及如何可以通过内源性疾病或外生环境因素的影响。高尔基-考克斯染色方法是在大脑中的神经元染色的随机样本的具有成本效益的，相对简单的装置。首先通过高尔基1在19世纪开发并由考克斯2改性，研究人员已经进一步改进这种技术多年来，以产生清晰，染色的神经元可被用于可视化和量化两者树突树形态和棘密度3,4 5，6，7，8，9。 对于染色的神经元的脑切片中的可视化的一个主要技术考虑是最大的切片厚度，这是由可用高倍率/高分辨率显微镜物镜的工作距离的限制。在60共同油浸物镜 - 100X范围上提供优异的分辨率，但他们的工作距离是通常不超过200微米的更大的限制。在200微米范围内切脑切片可能是足够的可视化可以被包含在此切片厚度内的某些神经元类型，在大脑皮质10，11，12，在的CA1区锥体细胞的浅层示例锥体神经元海马13，14，和颗粒细胞在海马15的齿状脑回。相对较长的神经元树突树，如大脑皮层的深层内锥体神经元，对于鼠标可以从细胞体16延伸大于800μm，提供了更大的挑战，因为大脑将需要在一个完美的角度被分段以包含整个树突树在200倍微米的切片。这甚至可能不是可行的，如果枝晶或任何其分支在延髓或尾方向延伸。虽然可以通过跟踪在多个相邻的脑切片中的神经元，以解决此限制，这种方法引入了在准确对准部分用于跟踪17显著的技术挑战。一个更实际的方法是可视化包含被以更大的厚度切脑切片内整个神经元。 我们在这里报告的技术染色400元以内 - 使用高尔基 - 考克斯方法小鼠的500微米厚的脑切片，并以可视化的MOrphology使用具有800微米的工作距离的高分辨率硅油浸物镜。我们描述了高尔基体考克斯浸渍和处理协议从最引用的现代协议之一在文献6改性。我们的与厚的脑切片方法提供了增加的标识被完全包含在节中的任何类型的神经元的概率的优点。除了主协议中，我们还提供独特的优点本两个变化:（1）高尔基体考克斯染色具有安装部分的表面上的甲酚紫染液中，为了限定脑区的边界，并确定的层大脑皮层，和（2）高尔基体考克斯染色与之前切片和最后的处理浸渍整个大脑的长期存储的中间冷冻步骤。

<table><tbody><tr><td>potassium dichromate</td><td>Fisher Scientific</td><td>P188-100</td><td>Hazardous</td></tr><tr><td>potassium chromate</td><td>Fisher Scientific</td><td>P220-100</td><td>Hazardous</td></tr><tr><td>mercuric chloride</td><td>Fisher Scientific</td><td>S25423</td><td>Hazardous</td></tr><tr><td>Whatman grade 1 filter paper</td><td>Fisher Scientific</td><td>1001-185</td><td></td></tr><tr><td>isoflurane</td><td>Pharmaceutical Partners of Canada</td><td>CP0406V2</td><td></td></tr><tr><td>20 mL scintillation vial</td><td>Fisher Scientific</td><td>03-337-4</td><td></td></tr><tr><td>sucrose</td><td>Bioshop Canada</td><td>SUC700.1</td><td></td></tr><tr><td>sodium phosphate monobasic</td><td>Sigma Aldrich</td><td>S5011-500G</td><td></td></tr><tr><td>sodium phosphate dibasic</td><td>Sigma Aldrich</td><td>S9390-500G</td><td></td></tr><tr><td>50 mL conical tube</td><td>Fisher Scientific</td><td>12-565-271</td><td></td></tr><tr><td>isopentane</td><td>Fisher Scientific</td><td>AC126470010</td><td>Also known as 2-methylbutane; hazardous</td></tr><tr><td>agar</td><td>Sigma Aldrich</td><td>A1296-100G</td><td></td></tr><tr><td>small weigh dish</td><td>Fisher Scientific</td><td>02-202-100</td><td></td></tr><tr><td>vibratome</td><td>Leica</td><td>VT1000 S</td><td></td></tr><tr><td>6-well tissue culture plates</td><td>Fisher Scientific</td><td>08-772-1b</td><td></td></tr><tr><td>mesh bottom tissue culture inserts</td><td>Fisher Scientific</td><td>07-200-214</td><td></td></tr><tr><td>paraformadelhyde (PFA), 16%</td><td>Electron Microscope Sciences</td><td>15710-S</td><td>Hazardous</td></tr><tr><td>ammonium hydroxide</td><td>Fisher Scientific</td><td>A669S-500</td><td>Hazardous</td></tr><tr><td>Kodak Fixative A</td><td>Sigma Aldrich</td><td>P7542</td><td></td></tr><tr><td>superfrost plus slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>CitroSolv clearing agent</td><td>Fisher Scientific</td><td>22-143-975</td><td></td></tr><tr><td>anhydrous ethyl alcohol</td><td>Commercial Alcohols</td><td>N/A</td><td></td></tr><tr><td>cresyl violet</td><td>Sigma Aldrich</td><td>C1791</td><td></td></tr><tr><td>permount</td><td>Fisher Scientific</td><td>SP15-100</td><td></td></tr><tr><td>upright microscope</td><td>Olympus</td><td>BX53 model</td><td></td></tr><tr><td>colour camera, 12 bit</td><td>MBF Biosciences</td><td>DV-47d</td><td>QImaging part 01-MBF-2000R-F-CLR-12</td></tr><tr><td>3D motorized microscope stage, controller and enoders</td><td>MBF Biosciences</td><td>N/A</td><td>Supplied and integrated with microscope by MBF Biosciences</td></tr><tr><td>4X microscope objective</td><td>Olympus</td><td>4x 0.16 N.A. UplanSApo</td><td></td></tr><tr><td>10X microscope objective</td><td>Olympus</td><td>10x 0.3 N.A. UPlan FL N&amp;nbsp;</td><td></td></tr><tr><td>30X microscope objective</td><td>Olympus</td><td>30x 1.05 N.A. UPlanSApo&amp;nbsp;</td><td></td></tr><tr><td>60X microscope objective</td><td>Olympus</td><td>60x 1.42 N.A. PlanAPO N</td><td></td></tr><tr><td>silicone immersion oil</td><td>Olympus</td><td>Z-81114</td><td></td></tr><tr><td>Neurolucida software</td><td>MBF Biosciences</td><td>Version 10</td><td></td></tr><tr><td>ImageJ software</td><td>U. S. National Institutes of Health</td><td>Current version</td><td>With the OME Bio-Formats plugin installed</td></tr><tr><td>Photoshop software</td><td>Adobe</td><td>version CS6</td><td></td></tr></tbody></table>

imaging neurons within thick brain sections using the golgi-cox method

神经细胞染色的高尔基 - 考克斯方法已使用超过两百年的脑组织样本中推进我们的神经元形态的理解。虽然它是从实用的观点考虑，优选以制备在最大厚度可能脑切片中，为了提高识别被完全包含单个区段内染色的神经元的概率，该方法是从由高工作距离技术的角度有限-magnification显微镜物镜。我们在这里报告的协议使用在小鼠脑切片的高尔基体考克斯方法，其是在500米微米厚的切割染色的神经元，并使用配有高分辨率30X 1.05 NA硅氧烷直立显微镜遍及这些区段的深度可视化的神经元油浸物镜，其具有800μm的工作距离。我们还报告这个协议可用于复染的表面的两个有用的变体安装的脑切片用甲酚紫染色尼斯尔，或以切片和最后的处理之前，冻结长期储存整个大脑。的主要协议和它的两个变体产生染色的厚的脑切片，在整个这充分神经元的树突树和枝晶棘可以可靠地可视化和量化。

此高尔基体考克斯染色方案和它的两个描述可选的变体可以被采用以400个内可视化的单个神经元 - 500微米厚的脑切片。使用10X物镜和在Z轴5层微米的步骤捕获的二维Z-突起的代表性图像的蒙太奇显示在图1:A1 - C1为冠状脑切片的大面积，其包括前扣带回皮层区域1和次级运动皮层18。切片在500微米切割。需要注意的是，其包括甲酚紫染色的协议变...

Watch this Scientific Journal Video about Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method at JoVE.com

Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

我们提出了一个协议，用于使用所述高尔基体考克斯染色法在厚的脑切片中，为了可视化与包含单个组织样品中的树突状长树木神经元。此协议的两种变型还提出涉及甲酚紫复染，和未加工的大脑进行长期存储的冻结。

厚脑切片使用高尔基考克斯方法中成像神经元

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within ...

imaging-neurons-within-thick-brain-sections-using-the-golgi-cox-method

Research

JoVE Journal

Neuroscience

18.2K 次观看.  University of Guelph. 我们提出了一个协议，用于使用所述高尔基体考克斯染色法在厚的脑切片中，为了可视化与包含单个组织样品中的树突状长树木神经元。此协议的两种变型还提出涉及甲酚紫复染，和未加工的大脑进行长期存储的冻结。 

视频: Imaging Neurons within Thick Brain Sections Using the Golgi-Cox Method

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

一个快速高分辨率的荧光成像的方法在半厚的脑片

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

科研

神经科学

Visualizing the Effects of a Positive Early Experience, Tactile Stimulation, on Dendritic Morphology and Synaptic Connectivity with Golgi-Cox Staining

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile stimulation is a positive early experience that mimics maternal licking and grooming in the rat. Exposing rat pups to this positive experience can be completed easily and cost-effectively by using highly accessible materials such as a household duster. Using a cross-litter design, pups are either stroked or left undisturbed, for 15 min, three times per day throughout the perinatal period. To measure the neuroplastic changes related to this positive early experience, Golgi-Cox staining of brain tissue is utilized. Owing to the fact that Golgi-Cox impregnation stains a discrete number of neurons rather than all of the cells, staining of the rodent brain with Golgi-Cox solution permits the visualization of entire neuronal elements, including the cell body, dendrites, axons, and dendritic spines. The staining procedure is carried out over several days and requires that the researcher pay close attention to detail. However, once staining is completed, the entire brain has been impregnated and can be preserved indefinitely for ongoing analysis. Therefore, Golgi-Cox staining is a valuable resource for studying experience-dependent plasticity.

This paper describes the procedures for tactile stimulation of rat pups and subsequent Golgi-Cox staining of neuronal morphology. Tactile stimulation is a positive experience that is administered in the perinatal period by stroking pups with a household duster. Golgi-cox staining is a reliable procedure permitting the visualization of entire neurons.

可视化的一个积极的早期经验，触觉刺激的影响，对树突状形态和突触连接与高尔基染色考克斯

To generate longer-term changes in behavior, experiences must be producing stable changes in neuronal morphology and synaptic connectivity. Tactile ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing patterns. The inclusion of biocytin/neurobiotin in the recording electrode permits post-hoc recovery of morphological details, which are necessary to determine the dendritic arborization and the regions targeted by the axons of the recorded neurons. However, given the presence of morphologically similar neurons with distinct neurochemical identities and functions, immunohistochemical staining for cell-type-specific proteins is essential to definitively identify neurons. To maintain network connectivity, brain sections for physiological recordings are prepared at a thickness of 300 &#181;m or greater. However, this thickness often hinders immunohistological postprocessing due to issues with antibody penetration, necessitating the resectioning of the tissue. Resectioning of slices is a challenging art, often resulting in the loss of tissue and morphology of the cells from which electrophysiological data was obtained, rendering the data unusable. Since recovery of morphology would limit data loss and guide in the selection of neuronal markers, we have adopted a strategy of recovering cell morphology first, followed by secondary immunostaining. We introduce a practical approach to biocytin filling during physiological recordings and subsequent serial immunostaining for the recovery of morphology, followed by the restaining of sections to determine the neurochemical identity. We report that sections that were filled with biocytin, fixed with paraformaldehyde (PFA), stained, and coverslipped can be removed and restained with a second primary antibody days later. This restaining involves the removal of the coverslip, the washing of sections in a buffer solution, and the incubation of primary and secondary antibodies to reveal the neurochemical identity. The method is advantageous for eliminating data loss due to an inability to recover morphology and for narrowing down the neurochemical markers to be tested based on morphology.

This protocol presents a method for the morphological recovery of neurons patched during electrophysiological recordings using biocytin filling and subsequent immunohistochemical postprocessing. We show that thick biocytin-filled sections that were stained and coverslipped can be restained with a second primary antibody days&#160;or months later.

对于神经化学标记用biocytin填充和加工部分的免疫染色

Electrophysiological recordings of cells using the patch clamp technique have allowed for the identification of different neuronal types based on firing ...

Large-scale Reconstructions and Independent, Unbiased Clustering Based on Morphological Metrics to Classify Neurons in Selective Populations

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a comprehensive survey of the morphological characteristics observed among a selective neuronal population. Combination of these techniques constitutes a novel approach for the collection and analysis of neuroanatomical data. Together, these techniques enable large-scale, and therefore more comprehensive, sampling of selective neuronal populations and establish unbiased quantitative methods for describing morphologically unique neuronal classes within a population.
The protocol outlines the use of modified rabies virus to selectively label neurons. G-deleted rabies virus acts like a retrograde tracer following stereotaxic injection into a target brain structure of interest and serves as a vehicle for the delivery and expression of EGFP in neurons. Large numbers of neurons are infected using this technique and express GFP throughout their dendrites, producing &#34;Golgi-like&#34; complete fills of individual neurons. Accordingly, the virus-mediated retrograde tracing method improves upon traditional dye-based retrograde tracing techniques by producing complete intracellular fills.
Individual well-isolated neurons spanning all regions of the brain area under study are selected for reconstruction in order to obtain a representative sample of neurons. The protocol outlines procedures to reconstruct cell bodies and complete dendritic arborization patterns of labeled neurons spanning multiple tissue sections. Morphological data, including positions of each neuron within the brain structure, are extracted for further analysis. Standard programming functions were utilized to perform independent cluster analyses and cluster evaluations based on morphological metrics. To verify the utility of these analyses, statistical evaluation of a cluster analysis performed on 160 neurons reconstructed in the thalamic reticular nucleus of the thalamus (TRN) of the macaque monkey was made. Both the original cluster analysis and the statistical evaluations performed here indicate that TRN neurons are separated into three subpopulations, each with unique morphological characteristics.

This protocol describes large-scale reconstructions of selective neuronal populations, labeled following retrograde infection with a modified rabies virus expressing fluorescent markers, and independent, unbiased cluster analyses that enable comprehensive characterization of morphological metrics among distinct neuronal subclasses.

基于形态学度量大规模重建和独立，公正聚类分类神经元选择性人群

This protocol outlines large-scale reconstructions of neurons combined with the use of independent and unbiased clustering analyses to create a ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

小鼠大脑皮层细胞组织的大尺度三维成像

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

将多重荧光 原 位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones -the sensory structures located at the distal tips of developing axons- execute this process. Studying the structure and dynamics of the growth cone is crucial to understanding axonal development and the interactions with the surrounding central nervous system (CNS) that enable it to form neural circuits. This is essential when devising methods to reintegrate axons into neural circuits following injury in fundamental research and pre-clinical contexts. Thus far, the general understanding of growth cone dynamics is primarily founded on studies of neurons cultured in two dimensions (2D). Although undoubtedly fundamental to the current knowledge of growth cone structural dynamics and response to stimuli, 2D studies misrepresent the physiological three-dimensional (3D) environment encountered by neuronal growth cones in intact CNS tissue. More recently, collagen gels were employed to overcome some of these limitations, enabling the investigation of neuronal development in 3D. However, both synthetic 2D and 3D environments lack signaling cues within CNS tissue, which direct the extension and pathfinding of developing axons. This protocol provides a method for studying axons and growth cones using organotypic brain slices, where developing axons encounter physiologically relevant physical and chemical cues. By combining fine-tuned in utero and ex utero electroporation to sparsely deliver fluorescent reporters along with super-resolution microscopy, this protocol presents a methodological pipeline for the visualization of axon and growth cone dynamics in situ. Furthermore, a detailed toolkit description of the analysis of long-term and live-cell imaging data is included.

In Situ Visualization of Axon Growth and Growth Cone Dynamics in Acute Ex Vivo Embryonic Brain Slice Cultures

This protocol demonstrates a straightforward and robust method to study in situ axon growth and growth cone dynamics. It describes how to prepare ex vivo physiologically relevant acute brain slices and provides a user-friendly analysis pipeline.

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

During neuronal development, axons navigate the cortical environment to reach their final destinations and establish synaptic connections. Growth cones ...

Rapid Golgi Stain for Dendritic Spine Visualization in Hippocampus and Prefrontal Cortex

Golgi impregnation, using the Golgi staining kit with minor adaptations, is used to impregnate dendritic spines in the rat hippocampus and medial prefrontal cortex. This technique is a marked improvement over previous methods of Golgi impregnation because the premixed chemicals are safer to use, neurons are consistently well impregnated, there is far less background debris, and for a given region, there are extremely small deviations in spine density between experiments. Moreover, brains can be accumulated after a certain point and kept frozen until further processing. Using this method any brain region of interest can be studied. Once stained and cover slipped, dendritic spine density is determined by counting the number of spines for a length of dendrite and expressed as spine density per 10 &#181;m dendrite.

The protocol describes a modification of the rapid Golgi method, which can be adapted to any part of the nervous system, for staining neurons in the hippocampus and medial prefrontal cortex of the rat.

厚脑切片使用高尔基考克斯方法中成像神经元

摘要

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一个快速高分辨率的荧光成像的方法在半厚的脑片

可视化的一个积极的早期经验，触觉刺激的影响，对树突状形态和突触连接与高尔基染色考克斯

树突分支中的海马区对小鼠的齿状回评

对于神经化学标记用biocytin填充和加工部分的免疫染色

基于形态学度量大规模重建和独立，公正聚类分类神经元选择性人群

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

小鼠大脑皮层细胞组织的大尺度三维成像

将多重荧光原位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

原位急性离体胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

快速高尔基染色用于海马体和前额叶皮层树突状脊柱可视化

厚脑切片使用高尔基考克斯方法中成像神经元

摘要

探索更多视频

此视频中的章节

一个快速高分辨率的荧光成像的方法在半厚的脑片

可视化的一个积极的早期经验，触觉刺激的影响，对树突状形态和突触连接与高尔基染色考克斯

树突分支中的海马区对小鼠的齿状回评

对于神经化学标记用biocytin填充和加工部分的免疫染色

基于形态学度量大规模重建和独立，公正聚类分类神经元选择性人群

使用高尔基Cox方法评估老年小鼠海马树突状复杂性

小鼠大脑皮层细胞组织的大尺度三维成像

将多重荧光 原 位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

原位 急性 离体 胚胎脑切片培养物中轴突生长和生长锥动力学的可视化

快速高尔基染色用于海马体和前额叶皮层树突状脊柱可视化

将多重荧光原位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

原位急性离体胚胎脑切片培养物中轴突生长和生长锥动力学的可视化