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。

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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

14.9K 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

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice. Fine motor coordination and balance can be assessed by the beam walking assay. The goal of this test is for the mouse to stay upright and walk across an elevated narrow beam to a safe platform. This test takes place over 3 consecutive days: 2 days of training and 1 day of testing. Performance on the beam is quantified by measuring the time it takes for the mouse to traverse the beam and the number of paw slips that occur in the process. Here we report the protocol used in our laboratory, and representative results from a cohort of C57BL/6 mice. This task is particularly useful for detecting subtle deficits in motor skills and balance that may not be detected by other motor tests, such as the Rotarod.

Deficits in fine motor coordination can be assessed with the balance beam test. Performance on the beam is quantified by the speed at which the beam is traversed and the number of times the mouse slips on the beam.

在小鼠体内的汽车平衡和协调的评估使用的平衡木

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

科研

神经科学

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

彗星试验测定DNA损伤的海马神经元

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

光遗传学的扰动激光照射半完好的神经活动<em&gt;果蝇</em&gt;幼虫运动

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

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 ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

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. ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

血管再生在中枢神经系统中使用的小鼠视网膜评估

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

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 ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

亚细胞钙荧光和生物发光成像<sup&gt; 2+</sup&gt;老年海马神经元

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...