Name: assessment of hippocampal dendritic complexity in aged mice using the golgi-cox method
Uploaded: 2017-06-22T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method at JoVE.com

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0900-1...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spines:+cellular+specializations+imparting+both+stability+and+flexibility+to+synaptic+function.">Dendritic spines: cellular specializations imparting both stability and flexibility to synaptic function.</a> Ann Rev Neurosci. 17, 341-371 (1994).</li><li>Leuner, B., Shors, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress,+anxiety,+and+dendritic+spines:+what+are+the+connections.">Stress, anxiety, and dendritic spines: what are the connections.</a> Neuroscience. 251, 108-119 (2013).</li><li>Harris, K. M., Fiala, J. C., Ostroff, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+at+dendritic+spine+synapses+during+long-term+potentiation.">Structural changes at dendritic spine synapses during long-term potentiation.</a> Philos Trans R Soc Lond B Biol Sci. 358 (1432), 745-748 (2003).</li><li>Kasai, H., Matsuzaki, M., Noguchi, J., Yasumatsu, N., Nakahara, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-stability-function+relationships+of+dendritic+spines.">Structure-stability-function relationships of dendritic spines.</a> Trends Neurosci. 26 (7), 360-368 (2003).</li><li>Das, G., Reuhl, K., Zhou, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Golgi-Cox+method.">The Golgi-Cox method.</a> Methods Mol Biol. 1018, 313-321 (2013).</li><li>Koyama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unending+fascination+with+the+Golgi+method.">The unending fascination with the Golgi method.</a> OA Anat. 1 (3), 24 (2013).</li><li>Pasternak, J. F., Woolsey, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+"selectivity"+of+the+Golgi-Cox+method.">On the "selectivity" of the Golgi-Cox method.</a> J Comp Neurol. 160 (3), 307-312 (1975).</li><li>Friedland, D. R., Los, J. G., Ryugo, D. 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Vibroslice NVSL &amp; Vibroslice NVSLM123 Available from: <a target="_blank" href="https://www.wpiinc.com/clientuploads/pdf/NVSL_NVSLM1_IM.pdf">https://www.wpiinc.com/clientuploads/pdf/NVSL_NVSLM1_IM.pdf</a> (2000)</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neurolucida 11.03. , (2017).</li><li>Sholl, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+organization+in+the+neurons+of+the+visual+and+motor+cortices+of+the+cat.">Dendritic organization in the neurons of the visual and motor cortices of the cat.</a> J Anat. 87 (4), 387-406 (1953).</li><li>Pillai, A. G., 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=Dendritic+morphology+of+hippocampal+and+amygdalar+neurons+in+adolescent+mice+is+resilient+to+genetic+differences+in+stress+reactivity.">Dendritic morphology of hippocampal and amygdalar neurons in adolescent mice is resilient to genetic differences in stress reactivity.</a> PLoS ONE. 7 (6), (2012).</li><li>Morley, B. J., Mervis, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spine+alterations+in+the+hippocampus+and+parietal+cortex+of+alpha7+nicotinic+acetylcholine+receptor+knockout+mice.">Dendritic spine alterations in the hippocampus and parietal cortex of alpha7 nicotinic acetylcholine receptor knockout mice.</a> Neuroscience. 233, 54-63 (2013).</li><li>Titus, A. 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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?...

<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 ...

The overall goal of this methodology is to properly Golgi stain brain tissue in order to examine and quantify dendritic morphology and complexity. This method can help answer key questions in neuroscience. Since Golgi staining only stains a small subset of cells, this technique could be used to trace the structure of neurons.The main advantage of this technique is that brain sections can be easily stained without risk to them drying out or falling off during any of the washing steps. Demonstrating the procedure will be Fred Kiffer, Tyler Alexander and Julie Anderson, graduate students in Dr.Allen's laboratory. Begin by immersing freshly harvested, unfixed rodent half brains in five milliliters or mercuric chloride-based solution.As mercuric chloride solution is light sensitive, cover the tube with foil. Store the samples in the dark at room temperature. The following day, slowly decant the solution into a temporary container before properly disposing of it in a biohazard waste container.Add five milliliters of fresh mercuric chloride solution and ensure that the brains are immersed. Return the samples to the dark at room temperature for 13 more days. After 14 days, prepare post-impregnation buffer by adding 30 grams of powered post-impregnation buffer to 90 milliliters of distilled or deionized water, DH2O.Fill each well of the six-well plate with 6.5 milliliters of the post-impregnation buffer, one well per brain. Then, remove the mercuric chloride solution from the brain tissue as before. Rinse the tissue with DH2O, then transfer the tissue to the six-well plates with post-impregnation buffer.Cover the plates with foil and store at room temperature in the dark. The next day, aspirate and replace the post-impregnation buffer. Re-cover the plate and store in a four degree Celsius fridge.Prepare two or three 12-well plates per brain by first labeling the plates on their lids with numbers in ascending order from left to right and up to down. Add the identification number of the brain sectioned. Pipette two milliliters of 1X PBS into each well of each plate, then set up the stage and turn on the vibratome.Then, put tape on the tissue block of the specimen holder and add a little cyanoacrylate glue on top. Remove a brain from the six-well plate and place it on a plastic or glass dish. Use a stainless steel double edged blade to cut the cerebellum caudally, making sure that the portion of the cerebellum remaining on the brain is flat.Then, place the flat surface of the remaining cerebellum on the glue. After the glue is dry, place the specimen holder with the tissue into the specimen bath. Place the specimen bath on the vibratome and cover the tissue sample in PBS.Then, attach the blade to the blade holder of the vibratome and slowly lower the blade into the specimen bath until it is fully submerged and slightly below the top of the tissue. Next, set the vibratome speed to seven, the amplitude to six and the cutting angle to 12 degrees. Start the vibration and cut 200-micron sections.Use a large paint brush to move the tissue sections from the specimen bath into each designated well of the 12-well plates. Continue to cut the tissue until the desired number of tissue sections is reached. After sectioning, use a transfer pipette to aspirate the PBS from the wells of the 12-well plate, then add two milliliters of PBS-T and wash for 30 minutes with agitation.Close to the end of the wash period, dilute three parts ammonium hydroxide solution with five parts DH2O. Then, after removing the PBS-T, add the diluted ammonium hydroxide solution. Protect from light using foil and stain the sections for 19 to 21 minutes.Then, after safely removing the ammonium hydroxide solution, add post-staining buffer. Protect with foil and stain for another 19 to 21 minutes. Finally, rinse the sections in PBS-T three times for five to 10 minutes each time.Mount the sections on 1%gelatin coated slides with the large paint brush. Allow them to dry for 20 to 30 minutes at room temperature, then place them in a Coplin Jar and leave overnight. Remove the slides from the Coplin Jar and place them in a plastic rack in the fume hood.Place in a staining dish containing 100%ethanol for five minutes. Dehydrate the sections through two further five-minute ethanol washes. Next, place the slides in a glass rack and use a metal handle to lower the rack into a glass staining dish containing 99%xylene.Wash the slides in two changes of xylene for five minutes each. After the time for the second wash has elapsed, remove the slides from the xylene one at a time and quickly cover the tissue with approximately 0.25 milliliters of mounting media. Next, take a slide cover and lay it over the media.Push any trapped air bubbles underneath the slide covers to the edge with a blunt object, such as the end of paint brush. Place the slides in an area out of direct sunlight and allow them to dry for two to three days. After drying, proceed to image acquisition using a microscope and analysis with the appropriate software.This bright field image shows a Golgi-stained neuron that meets the criteria for imaging and further analysis. All three spine types can easily be seen. Staining is consistent across the entire length of the dendrite and the dendrite is isolated from other neurons.This figure shows that Golgi stained neurons within the Dentate Gyrus showed decreased length at the fourth and sixth order following 5-fluorouracil treatment compared to the saline-treated group. Golgi staining can be used to detect significant differences in spine density. In the CA1 apical pyramidal dendrites, 5-fluorouracil administration to live mice significantly decreased spine density.In the basal dendrites, there were no significant changes in the overall density of spines. Sholl Analysis of CA1 apical pyramidal dendrites revealed that 5-fluorouracil treatment significantly decreased dendritic arborization at distances between 40 to 160 microns away from the soma. In the basal dendrites, the 5-fluorouracil treatment decreased the dendritic arborization at 30 to 110 microns away from the soma.Once mastered, this technique, accounting for both the Golgi staining process and mounting, cleaning and covering can be done in three hours per brain if performed properly. After its development, this technique paved the way for researchers in the field of neurobiology to explore neuroanatomical connections in a murine model. While attempting this procedure, it's important to remember to keep the sections in ammonium hydroxide solution for only 19 to 21 minutes as to not overstain the neurons.After watching this video, you should have a good understanding of how to properly stain, section and rinse brain tissue. Don't forget that working with the mercuric chloride-based solution, ammonium hydroxide solution and xylene can be extremely hazardous, so take precautions and wear lab gloves and work in a fume hood when performing this procedure.

Research

JoVE Journal

Neuroscience

Assessment of Motor Balance and Coordination in Mice using the Balance 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

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

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

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

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

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

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

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

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

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

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

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

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

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

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

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 ...