这项工作得到NIH P20 GM109005（ARA）和转化神经科学中心IDeA计划奖P30 GM110702的试点资助。

实验按照UAMS机构动物护理和使用委员会批准的道德标准进行。 动物和5-Fu注射模式<ol><li>购买6个月大的雄性C57Bl6 / J野生型小鼠，并将其置于恒定的12小时光/黑暗循环下，直至达到1岁。 </li><li>在0.9％无菌盐水中稀释5-Fu。使用60 mg / kg作为每只小鼠所需的剂量。 </li><li> 腹腔注射5-Fu（每周一次，持续三周）。 <ol><li>每天在同一时间内进行腹膜内注射。例如，在0900-1200小时之间。 </li></ol></li><li>在最后一次注射后30天安乐死动物并取出大脑。 </li></ol> 2.安乐死程序和脑提取<ol><li>禁止啮齿动物抓住尾巴的基部。另一方面，将拇指或第一根手指放在第二位e是啮齿动物颈部的基础。迅速对啮齿动物的脖子施加压力，向前推，另一只手抓住尾巴向后拉。 </li><li>用一只手握住啮齿动物，另一只握住大手术剪刀。将啮齿动物的脖子放在两个刀片之间，并迅速斩首。 </li><li>拿着切断的鼠标头，用精细的剪刀剪掉头骨上方的毛皮，直到眼睛。接下来，将剪刀的尖端放在每个眼窝中，轻轻地关上剪刀。 </li><li>使用弹簧剪将头骨切割到小脑的左侧和右侧。 </li><li>使用镊子将小脑周围的颅骨的部分直接抬起和关闭。 </li><li>使用弹簧剪刀沿着头骨的中心线切割。 </li><li>使用镊子将头骨的剩余部分抬离大脑。 </li><li>使用刮铲和探头将大脑舀出到所需的容器中。使用不锈钢teel刀片，沿着中心位置切割每个脑。在右半球执行以下Golgi-Cox方法。 </li></ol> 3.高尔基染色和组织制备<ol><li>将新鲜收获的半脑浸入10 mL锥形管中的5 mL氯化汞基溶液（试剂盒中的溶液A）。氯化汞溶液对光敏感;用箔覆盖管。将样品在室温下保存在黑暗中。 注意:在材料表中列出的试剂盒中提供了溶液A。警告！溶液A（也称为氯化汞溶液）含有毒性试剂如重铬酸钾，氯化汞和铬酸钾。戴防护手套，并在通风橱中工作。 </li><li> 1天后，将溶液缓慢倾析成临时容器（如大型称重船），并将其处理在生物危害废物容器中。将大脑（仍在原来的10 mL锥形管中）浸入5 mL氯化汞溶液，并将样品在室温下返回黑暗13天以上。 </li><li> 向90 mL蒸馏水或去离子水（dH 2 O）中加入30 g浸渍后缓冲液。向6孔板的每个孔中填充6.5mL后浸渍缓冲液，每个大脑一个孔 注意:在材料表中列出的试剂盒中提供了浸渍后缓冲液。 <ol><li>在氯化汞溶液中14天（总共）后，用dH 2 O冲洗组织，然后将其转移到具有后浸渍缓冲液的6孔板中。用箔盖住板，并在室温下将其存放在黑暗中。 </li></ol></li><li>浸泡1天后取出浸渍后的缓冲液，并用新鲜的浸渍后缓冲液更新;在室温下在黑暗中储存1天。如果需要，存储在4°C最多1个月22 。 </li></ol>分段<ol><li>在其盖子上标记12孔板，数字从左到右和从上到下按升序排列。 </li><li> 如果切断整个大脑，每脑准备两个或三个12孔板。 <ol><li>标记每个12孔板盖的顶部与脑部分的识别号码。 </li><li>将2 mL 1x PBS吸取到每个板的每个孔中。 </li></ol></li><li> 设置舞台，打开vibratome;确保容器中有足够的1x PBS以覆盖组织。 <ol><li>切割两片塑料石蜡膜并折叠两次。将它们放在样品支架旋钮的孔上，以防止1x PBS泄漏到计数器上。 </li><li>将胶带放在样品架的组织块上，然后将氰基丙烯酸酯胶粘剂（见表格）放在上面。 </li></ol></li><li> 从6孔板中取出大脑并将其放在塑料或玻璃皿上。使用不锈钢钢双刃刀片切断大约一半的小脑。 <ol><li>头尾切小脑。确保剩下的小脑部分是均匀的。 </li></ol></li><li> 立即将剩余小脑的平坦表面放在胶上。 注意:组织（大脑皮层）的背部部分应相对于刀片面向左或右。以前包含嗅球的前端应朝上。 <ol><li>等待至少几分钟，以确保胶水完全干燥。 </li></ol></li><li> 当将大脑保持在适当位置的胶水正在干燥时，将1x PBS倒入样品池中。胶水干燥后，将样品架放入样品池中。 <ol><li>如果组织样本没有完全覆盖，则将更多的1×PBS倒入样品池中。 </li></ol></li><li>将刀片连接到振动片的刀架上，慢慢地l将刀片放入样品池中，直到其完全浸没在1x PBS中。继续降低刀片，直到其稍低于组织的顶部。 </li><li> 将振动速度设置为7，振幅为6，切割角度为12°（参见表的材料为vibratome型号）。启动振动并切割200微米的部分23 。 <ol><li>使用大型画笔将组织切片从样品池移动到12孔板的每个指定的孔中。 </li></ol></li><li>继续切割组织直到达到所需数量的组织切片。 </li></ol>后染色<ol><li>对于以下每个染色步骤和冲洗液，每孔使用2 mL所示溶液。使用电动移液器填料（参见表格）将溶液转移到孔中。使用转移移液器（参见表格）将溶液从井中转移到适当的废物中容器。 </li><li>在0.01M PBS-T（加入3.0mL洗涤剂（参见表格物质））至1,000mL 0.01M PBS中的一个30分钟洗涤中冲洗切片。 </li><li> 稀释氢氧化铵溶液（注意事项）立即使用前用dH 2 O容积3:5。将稀释的氢氧化铵溶液中的切片染色19-21分钟。 <ol><li>用箔保护光敏氢氧化铵溶液。 注意:溶液中的氢氧化铵浓度为〜10％，被归类为"危险"。如果氢氧化铵溶液接触皮肤，可能会产生灼伤。吸入可能引起呼吸道刺激。戴防护手套，并在通风橱中工作。 </li></ol></li><li>染色后染色缓冲液中的切片（在500 mL dH 2 O中加入90 g试剂D）19-21分钟。后染色缓冲液是光敏感的;用箔22保护它。 </li><li>冲洗三部分，洗涤5-10M PBS-T。 </li></ol> 6.安装，清洁和覆盖<ol><li>用大画笔在1％明胶涂层的载玻片上安装切片。让它们在室温下干燥20-30分钟，然后将其置于Coplin罐中过夜。 </li><li>从Coplin罐中取出戴手套的幻灯片，并将它们放在塑料支架中（参见表格）。将塑料架置于含有100％乙醇的染色盘（见表格）中。用三次洗涤5分钟使其脱水。 </li><li>每次在99％二甲苯中洗涤载玻片两次，每次5分钟。将载玻片放置在玻璃架中，并将机架放入含有金属把手的二甲苯的玻璃染色皿中（参见表格）。 注意:注意，二甲苯吸入有害，如果接触皮肤，会引起刺激。它在液体和蒸气状态下是高度易燃的。戴防护手套，并在通风橱中工作。 </li><li> 从xyl中取出幻灯片一次一个，并用〜0.25 mL的安装介质快速覆盖组织（参见材料表）。接下来，把幻灯片盖子放在媒体上。 <ol><li>将滑盖的下方的任何被困的气泡用钝器（如画笔的末端）推到边缘。 </li></ol></li><li>将幻灯片放置在远离阳光的地方，让它们干燥2-3天。 </li><li>继续使用显微镜获取图像，并使用适当的软件进行分析。 </li></ol> 7.获取图像堆栈<ol><li>打开成像程序（参见材料表 ）。将幻灯片放置在显微镜的舞台上。在程序顶部的菜单上，单击"获取"，然后单击"实时图像"。 </li><li>将显微镜设置为10X。识别偏好的神经元，然后将光标（如红色X）移动到索马里的中心。左键单击设置参考点。 </li><li>将显微镜设置为40X。从程序顶部的菜单中单击"移动"，然后单击"到参考点"。 </li><li> 开始创建图像堆栈，手动滚动组织上方的细微物体，直到它稍微偏离焦点。 <ol><li>点击程序右下角"图像采集"标题下的"设置顶部"。 </li><li>稍稍滚动一下纸巾，直到它略微偏离焦​​点，然后点击"图像采集"下的"设置底部"。接下来，点击"获取图像堆栈"。 </li></ol></li><li> 获取图像堆叠后，在出现的窗口中输入图像文件所需的名称。另存为"MBF Ascii文件"。 <ol><li>出现提示时，选择文件类型为"MBF JPEG2000堆栈文件"，然后单击"保存"。 </li></ol></li></ol>神经元追踪<ol><li>在工具栏r在菜单下方，单击标题为"轮廓名称"的框。从下拉菜单中选择"Soma"。 </li><li>手动追踪索马然后右键单击并选择"完成单元格主体"，当跟踪完成。 </li><li>选择"AutoNeuron"以显示另一个标题为"AutoNeuron Workflow"的标签。 </li><li> 在"配置类型"下，选择"新建"。设置参数，然后单击"下一步"以显示"Soma Detection"子标题。 <ol><li>要设置参数，请选择"Brightfield"。接下来，在"最大工艺直径"下，单击"开始测量"。使用光标，转到树枝的底部并手动设置直径。 </li></ol></li><li>在请求用户跟踪整个图像的窗口中单击"是"。手动追踪索马单击"下一步"，打开"种子放置"子标题。 </li><li>点击"验证种子"s"，然后点击"下一步"提出"神经元重建"副标题。 </li><li>在"神经元"下，点击"互动"。执行交互式跟踪，遵循树突程序的方向，右键单击以完成程序跟踪的突出显示区域。跟踪所有树突后，单击"下一步"。 </li><li>出现新窗口后，保存具有所需标题的新配置。点击"保存"。 </li></ol> 9.分析<ol><li>打开分析程序（参见材料表）。 </li><li>单击顶部菜单中的"文件"，然后单击"打开数据文件"。选择感兴趣的文件。 </li><li>在分析小标题下，选择"分析"。 </li><li>在"Sholl Analysis"窗口中，将起始半径设置为10μm。在"分析"下，单击"树枝"和"分支订单"框。 </li><li>右对齐ick新打开的窗口，然后单击"Excel导出"。点击"保存"。 </li><li>在分析下，点击"分支结构分析"。选择"所有可能的分析"。 </li><li>右键单击新打开的窗口，然后单击"Excel导出" 24 。 </li></ol>

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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypobaric+hypoxia-induced+dendritic+atrophy+of+hippocampal+neurons+is+associated+with+cognitive+impairment+in+adult+rats.">Hypobaric hypoxia-induced dendritic atrophy of hippocampal neurons is associated with cognitive impairment in adult rats.</a> Neuroscience. 145 (1), 265-278 (2007).</li><li>Groves, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Fluorouracil+chemotherapy+upregulates+cytokines+and+alters+hippocampal+dendritic+complexity+in+aged+mice.">5-Fluorouracil chemotherapy upregulates cytokines and alters hippocampal dendritic complexity in aged mice.</a> Behavioral Brain Research. 316, 215-224 (2017).</li><li>Risher, W. C., Ustunkaya, T., Singh Alvarado, J., Eroglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Golgi+analysis+method+for+efficient+and+unbiased+classification+of+dendritic+spines.">Rapid Golgi analysis method for efficient and unbiased classification of dendritic spines.</a> PloS One. 9 (9), (2014).</li><li>Kaufmann, W. E., Moser, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+anomalies+in+disorders+associated+with+mental+retardation.">Dendritic anomalies in disorders associated with mental retardation.</a> Cerebral cortex. 10 (10), 981-991 (2000).</li><li>Kulkarni, V. A., Firestein, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dendritic+tree+and+brain+disorders.">The dendritic tree and brain disorders.</a> Mol Cell Neurosci. 50 (1), 10-20 (2012).</li></ol>

作者宣称他们没有竞争的经济利益。

与更现代的技术相比，高尔基Cox方法具有几个优点，使其成为检查脊柱形态的首选方法:1）染色可用于基本上任何组织，2）基本的光学显微镜设置是所有需要的获得基于高尔基体的图像，3）高尔基Cox成像比共焦成像更快; 4）高尔基染色切片比荧光标记的样品长达数月至数年。即使有这些优点，高尔基Cox方法仍然有一定的局限性。首先，整个过程非常耗时，需要几周的染色，然后分析图像。如本协...

<html>树突状体是接收和处理突触前输入的神经元的最大部分1 。它们的树突过程具有复杂的几何形状，其中近端分支具有比远端分支更大的直径。当树突发育时，它们在称为树突状树突的过程中与其他神经元形成几个连接。这种分支的程度和模式决定了树突可充分处理的突触输入量2 。 树突状晶化是活性依赖性可塑性和神经元回路适当发育的必需过程。延伸，收缩，分支和突触发生是复杂的过程，包括内在遗传程序和外在因素的影响。海马内神经元树突的形态和分支变化涉及认知和记忆形成3，4 ，树突状复杂性的改变与病理生理和行为变化有关5 ，异常与多种疾病状态有关，包括脆性X综合征和唐氏综合征6 。 树枝状刺是树突状乔木的特殊亚细胞层，在中枢神经系统内接受兴奋性输入。有三种形态类型的树突棘，每个类的名称根据其大小和形状:1）蘑菇棘，具有比其他刺7更复杂的突触后密度，具有更多的谷氨酸受体; 2）粗短的刺，缺乏茎;和3）薄刺，由长而窄的茎和球状头组成。树突状体积部分地用于限定它们，其中薄脊通常较小（0.01μm3 </sup&gt;）与蘑菇刺（0.8μm3） 9,10比较 。脊椎稳定成熟。例如，薄的脊椎在几天之后缩回或发展成蘑菇刺。或者，蘑菇棘相对稳定并且可以长时间存活。神经元连接的强度被认为是基于刺的数量和/或其体积11,12,13 。 古典高尔基染色方法及其更现代的变化对于检查树突棘形态和密度都是有用的。高尔基染色的一个独特之处在于它随机染色了约5％的总神经元，这允许追踪个体神经元14,15 。虽然高尔基甲基的确切机制od染色单个神经元尚未知，该方法的原理是基于铬酸银（Ag 2 CrO 4 ）的结晶16,17 。高尔基方法有三种主要类型:快速高尔基体，高尔基体细胞，高尔基 - 科罗奇18，19 。所有这三种方法从铬盐的初始孵化阶段开始数天至数月，但它们之间存在一定的关键差异。快速高尔基体在第一步使用四氧化锇，而高尔基Kopsch包括多聚甲醛。随后在1-2％硝酸银溶液中孵育约7天，在快速高尔基体和高尔基体系中进行染色。高尔基Cox法使用氯化汞和重铬酸钾代替硝酸银，浸渍时间为2-4周。然后将组织切片并快速置于稀释的氨中溶液，然后用照相定影剂除去盐。在三种类型中，高尔基Cox方法被认为是在没有太多背景干扰下染色树枝状树枝的最佳方法，部分原因是因为晶体表面不会发生在组织表面（不像快速高尔基法） 17，20，21 。 本方法是商业高尔基染色试剂盒提供的方案的轻微变化，并且设计用于评估相对低剂量的5-Fu如何影响树突状态特征和脊柱密度。获得的任何数据可以进一步了解化学治疗如何影响神经元电路。

<table><tbody><tr><td>superGolgi Kit&amp;nbsp;</td><td>Bioenno Lifesciences</td><td>30100</td><td>&amp;nbsp;Contains hazardous materials.&amp;nbsp;</td></tr><tr><td>PBS 10x powder concentrate</td><td>Fisher&amp;nbsp;</td><td>BP665-1</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>9002-93-1</td><td></td></tr><tr><td>Permount&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>SP 15-100</td><td></td></tr><tr><td>Slide cover&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>12-546-14</td><td></td></tr><tr><td>7 mL Transfer pipette&amp;nbsp;</td><td>Globe Scientific&amp;nbsp;</td><td>135030</td><td></td></tr><tr><td>10 mL Falcon tubes&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>352099</td><td></td></tr><tr><td>Foil&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>01-213-105</td><td></td></tr><tr><td>12-well plate&amp;nbsp;</td><td>BD Biosciences&amp;nbsp;</td><td>353043</td><td></td></tr><tr><td>200 proof Ethanol&amp;nbsp;</td><td>Pharmco-AAPER</td><td>111000200</td><td></td></tr><tr><td>Xylene&amp;nbsp;</td><td>Acros Organics&amp;nbsp;</td><td>1330-20-7</td><td>Hazardous.&amp;nbsp;</td></tr><tr><td>Permabond 200</td><td>Permabond LLC</td><td>GF2492</td><td></td></tr><tr><td>25 mL serological pipette</td><td>Sigma</td><td>SIAL1489</td><td></td></tr><tr><td>Parafilm</td><td>Midsci</td><td>HS234526C&amp;nbsp;</td><td></td></tr><tr><td>Vibratome&amp;nbsp;</td><td>World Precision Instruments&amp;nbsp;</td><td>NVSLM1</td><td></td></tr><tr><td>C57Bl/6 Male Mice&amp;nbsp;</td><td>The Jackson Laboratory&amp;nbsp;</td><td>000664</td><td></td></tr><tr><td>Axio Imager 2</td><td>ZEISS</td><td>Multiple components, see website for details.&amp;nbsp;</td><td></td></tr><tr><td>AxioCam MRc Camera</td><td>ZEISS</td><td>426508-9902-000</td><td></td></tr><tr><td>Staining Dish , Green</td><td>Tissue-Tek</td><td>62541-12</td><td></td></tr><tr><td>Staining Dish Set&amp;nbsp;</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>70312-20</td><td></td></tr><tr><td>Motorized Pipet Filler&amp;nbsp;</td><td>Fisher&amp;nbsp;</td><td>03-692-168</td><td></td></tr><tr><td>Neurolucida&amp;nbsp;</td><td>mbf Bioscience&amp;nbsp;</td><td></td><td></td></tr><tr><td>Neurolucida Explorer&amp;nbsp;</td><td>mbf Bioscience&amp;nbsp;</td><td></td><td></td></tr><tr><td>Prism&amp;nbsp;</td><td>GraphPad</td><td></td><td></td></tr></tbody></table>

assessment of hippocampal dendritic complexity in aged mice using the golgi-cox method

树枝状突起是包含兴奋性突触的神经元树突状突起。海马内神经元树突的形态和分支变化涉及认知和记忆形成。有几种高尔基染色方法，所有这些方法都可用于确定树突状乔木的形态特征并产生清晰的背景。目前的高尔基Cox方法（商业高尔基染色试剂盒提供的方案略有变化）被设计用于评估相对较低剂量的化疗药物5-氟尿嘧啶（5-Fu）如何影响树突状形态，脊柱数量以及海马内部植树的复杂程度。 5-Fu以区域特异性方式显着调节树突状复杂性并降低整个海马的脊柱密度。数据显示，高尔基染色法对CA1，CA3和海马齿状回（DG）中的成熟神经元进行了有力的染色。该协议报告每个步骤的细节，以便其他研究人员能够以高质量的结果和最少的故障排除，可靠地在整个大脑中染色组织。

使用市售成像软件量化5-Fu处理对高尔基染色脑切片海马树突状结构和复杂性的影响。在追踪后，使用Sholl分析和树突复合指数（DCI）分析树突状树枝状结构，脊柱密度和脊柱形态。 Sholl分析是一种定量分析方法，可用于确定树枝状心轴形态25 。从soma开始，距离彼此相隔10微米的圆圈覆盖树突状迹。枝晶的长度取决于枝晶交叉的圆数。分支点分析，一种确?...

Watch this Scientific Journal Video about Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method at JoVE.com

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

这里我们详细介绍一种高尔基体Cox协议。这种可靠的组织染色方法允许对海马中的细胞结构和整个大脑进行高质量的评估，同时进行最少的故障排除。

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 ...

assessment-hippocampal-dendritic-complexity-aged-mice-using-golgi-cox

Research

JoVE Journal

Neuroscience

15.0K 次观看.  University of Arkansas for Medical Sciences. 这里我们详细介绍一种高尔基体Cox协议。这种可靠的组织染色方法允许对海马中的细胞结构和整个大脑进行高质量的评估，同时进行最少的故障排除。 

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

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

突触改变老化过程中淀粉样蛋白病理研究从大鼠和转基因小鼠海马急性片的制备

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

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 ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

从成年小鼠海马齿状回的剖析

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

生物学

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 ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

海马神经元活动的可视化免疫组化空间后学习神经发育障碍的小鼠模型

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

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

The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within histological brain samples. While it is preferable from a practical perspective to prepare brain sections at the greatest thickness possible, in order to increase the probability of identifying stained neurons that are fully contained within single sections, this approach is limited from a technical perspective by the working distance of high-magnification microscope objectives. We report here a protocol to stain neurons using the Golgi-Cox method in mouse brain sections that are cut at 500 &#956;m thickness, and to visualize neurons throughout the depth of these sections using an upright microscope fitted with a high-resolution 30X 1.05 N.A. silicone oil-immersion objective that has an 800 &#956;m working distance. We also report two useful variants of this protocol that may be employed to counterstain the surface of mounted brain sections with the cresyl violet Nissl stain, or to freeze whole brains for long-term storage prior to sectioning and final processing. The main protocol and its two variants produce stained thick brain sections, throughout which full neuron dendritic&#160;trees and dendrite spines may be reliably visualized and quantified.

We present a protocol for using the Golgi-Cox staining method in thick brain sections, in order to visualize neurons with long dendritic&#160;trees contained within single tissue samples. Two variants of this protocol are also presented that involve cresyl violet counterstaining, and the freezing of unprocessed brains for long-term storage.

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 ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

成人希马神经前体细胞的乳化电路特定调节

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

从成人和老化小鼠的河马切片中尽量减少低氧

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

从多萨尔海马的急性切片准备全细胞记录和神经元重建在成人小鼠的登特陀螺

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

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.