Name: assessment of dendritic arborization in the dentate gyrus of the hippocampal region in mice
Uploaded: 2015-03-31T13:00:00.000+00:00
Duration: 10 min 55 s
Description: Watch this Scientific Journal Video about Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice at JoVE.com

This research was supported by grants from the LuMind Foundation, Research Down Syndrome, and the Alzheimer&#8217;s Association (AS). CP was partially supported by a faculty development grant from the College of Nursing and Health Professions at Arkansas State University.

注:实验按照批准委员会在动物研究在退伍军人事务帕洛阿尔托医疗保健系统的道德标准进行的。 1.高尔基 - 考克斯染色<ol><li>脑提取和染色<ol><li>第1天，通过放血安乐死前，深深地麻醉小鼠100毫克/千克氯胺酮和10毫克/公斤甲苯噻嗪。 </li><li>小心地取出颅骨，并剖析了大脑。 <ol><li>首先去除皮肤上的头骨的顶部，放置一个弯曲剪刀对小脑的顶部并轻轻切穿平行颅盖到中央沟与剪刀的尖端指向向外，朝着朝向嗅球的方向。两侧重复此过程。 </li><li>用手术pincet解除颅骨，同时把它朝嗅球，并打破它。然后，翻转头骨倒过来，用一个接通过视神经切神经和放松大脑。最后，使用挑到大脑从脊髓在枕骨大孔的水平分开。 </li></ol></li><li>立即浸入脑在15毫升市售，未稀释的高尔基体考克斯溶液。存储在大脑中加盖，将20ml玻璃瓶高尔基溶液在室温下在黑暗中。 </li><li>第2天，缓缓倾出溶液，并用15毫升新鲜高尔基考克斯溶液替换。回顾一下含有大脑的瓶子，并留下静置在黑暗中另一个9天。 </li></ol></li><li>切片和嵌入<ol><li> 11日当天，将大脑中30％的蔗糖溶液，准备在DDH 2 O的脱水。 12小时后改变与新配制的30％的蔗糖溶液。孵育3天30％蔗糖溶液的大脑在4℃。 </li><li>在第14天，填的vibratome的贮存用30％的蔗糖溶液，直到刀片被覆盖。设置速度1米米/秒的0.95毫米幅度。 </li><li>吸干大脑用Kimwipe以除去过量的水分并用剃刀刀片切冠除去小脑。 </li><li>使用强力胶牢固地装入整个大脑上的vibratome平台，并允许它为2-4分钟，进行设置。插入与附着脑平台进入储和叶片调整到大脑的顶部。 </li><li>使用的vibratome，切片脑内成150微米厚的切片在室温下，记忆每第三大脑后改变刀片。 </li><li>拿起从储存的部分用小刷子尖和浸入成含有0.3％明胶在双蒸2 O制成的陪替氏培养皿而部分残留在培养皿中，添加0.5毫升0.3％明胶的每个幻灯片上，并用刷子来传播和涂覆的Superfrost +幻灯片。 </li><li>轻轻拿起浸渍部分从培养皿并将其放置到胶凝幻灯片。通过定向脑切片触摸它们只在其两侧，而不是顶部。 </li><li>经过约10分钟，涂上30％蔗糖的章节之前组织完全干燥。然后空气干燥，于黑暗处准备的滑动的滑动架3天。 </li><li>稀释市售10倍显影液至1x使用双蒸2 O.浸泡在显影液7-10分钟的幻灯片。冲洗玻片3次，双蒸2 O. </li></ol></li><li>脱水<ol><li>用蒸馏水双蒸2 O制备的20％，30％，40％，50％，60％，70％，80％，90％和95％的乙醇溶液</li><li>浸泡的幻灯片中越来越高的乙醇溶液，每次10分钟。 </li><li>浸泡的幻灯片在100％的乙醇溶液，每次10分钟，3次。 </li><li>浸泡的幻灯片在100％二甲苯中连续3次，每次5分钟，保持部分在最终溶液中，直到它们盖滑落。 </li><li>将DPX（二正丁酯，邻苯二甲酸酯在二甲苯）的安装介质（AB的大方量出1-1.5毫升）用塑料吸管转移每张幻灯片上。小心地将盖玻片，以避免气泡。 </li><li>空气干燥盖滑脑切片水平3天。 </li></ol></li></ol> 2. Doublecortin染色<ol><li>深深麻醉（见步骤1.1.1）的动物。 </li><li>用新鲜配制的4％多聚甲醛磷酸盐缓冲液12发进行心脏灌注。 </li><li>提取大脑（见1.1.2），并存储于45ml的4％多聚甲醛在4℃下过夜上的摇床上。 </li><li>大脑转移到30％蔗糖溶液，并等待完整脱水48小时，在4℃。从蔗糖取出大脑，并直接将它们放在置于干冰上的铜块。 </li><li>填具有最佳切割温度（OCT）化合物的块，并使用嗅球作为地标标记的脑的定向。 </li><li>使用低温恒温器，切割70微米厚的切片在-20ºC，并把它们在冷冻保护剂溶液（25％乙二醇，25％的甘油，在0.05M磷酸钠缓冲液，pH 7.4）中，并保持在-20℃直到使用。 </li><li>使50％的冷冻保护剂和50％的甲醇的溶液。向此溶液中添加0.5％过氧化氢（体积/体积）。孵育浮动切片30分钟，在室温下进行。 </li><li>在37ºC的TBS（Tris缓冲盐水）30-40分钟暖段通过将它们放置在一个组织学烘箱。 </li><li>孵育0.3％的Triton和3％正常马血清的章节之前，免疫染色45分钟，在室温下进行。孵育4ºC的部分一夜之间在DCX抗体稀释1 3毫升的解决方案:500 TBS与0.3％的Triton和1％的正常马血清。 </li><li>翌日洗部分连续3次的TBS（各10分钟）。 </li><li>孵育用生物素化的马抗山羊部分（1:200在TBS​​中）为2小时，在室温下进行。在TBS中洗部分连续3次（每次10分钟）。 </li><li>孵育吨下摆与ABC精简版（1:1,000）1.5小时，在室温下进行。 </li><li>加入5微升30％的H 2 O 2至10毫克DAB的溶解于15ml的1M的Tris-HCl（pH值7.6）的（3,3"二氨基联苯胺）溶液1片剂。在该溶液中5分钟，立即孵育部分。 </li><li>洗涤他们用冰冷的TBS三次随后在TBS中洗涤一次，在室温下使反应终止。 </li><li>脱水用一系列乙醇溶液（50％，60％，70％，80％，90％，95％，100％，每5分钟），明确在二甲苯的部分，然后用覆盖的DPX滑移如在步骤1.3。 </li></ol> 3.可视化<ol><li>继部分完全干燥，去除用锋利的刀片幻灯片上面多余的DPX，并把他们放在显微镜扫描阶段。该系统由装备有扫描阶段，控制杆，和一个彩色12位相机的显微镜的。 </li><li>启动该程序。打开一个新的数据文件。 </li><li>地方通过点击鼠标指针在任何地方在屏幕上的参考点。这将激活所有从程序窗口的工具面板的图标。 </li><li>点击工具栏上的"摇杆自由"图标，然后用操纵杆来定位海马齿状回的第一部分。 </li><li>在程序窗口的"工具"选项卡，选中"序列部门经理"。点击在窗口的左下角的"新科"图标。这将打开"串行科设置"窗口。 </li><li>选择的串行部分设置窗口，然后输入包含海马区切片的总数。选择评估间隔。输入部分切割厚度（70微米的DCX和150微米的高尔基染色切片）。 </li><li>首先点击工具栏中的"写意轮廓图"图标，跟踪齿状回区。概述齿状颗粒细胞层。&lt;/ li&gt; <li>从"放大"菜单中选择"100X"。添加一滴浸油的部分和切换目标100X。在找到这一目标的齿状颗粒细胞体和树突。 </li><li>专注于一个选定的神经元，然后单击工具栏上的"神经元跟踪"图标。跟踪齿状颗粒细胞体的周长。当跟踪完成后，单击鼠标右键，选择"完成胞体"。 </li><li>开始的x，y和z方向手动跟踪的树突和使用操纵杆和z马达旋钮按照每个分支。在分叉或三叉节点，选择在右键下拉菜单中选择相应的选项。跟踪每个源于这些节点的分支。在每个分支末端单击鼠标右键，从下拉菜单中选择"结束"。 </li><li>在软件窗口中使用箭头键图标随机选择另一齿状颗粒细胞，并按照同样的程序DESCRIBED步骤3.9-3.10。保存整个跟踪。 </li><li>启动神经跟踪软件。 </li><li>从实验组的第一鼠标打开第一NRX数据文件。追加实验组的第二只老鼠的NRX文件。继续下去，直到该组所有的NRX文件被追加。 </li><li>在分析选项卡，选择"范在-图"。这将打开风扇在分析窗口。查马克"树突"，然后单击显示，描绘树突的长度和分支模式这组小鼠（ 图1）。 </li></ol> 4. EDFI 注意:此方法使用户通过每个场的z轴快速记录大量图像并生成包含所有的聚焦像素整个z轴2D图像。其结果将是一个包含从收集到的图像都聚焦像素的合成图像。 <ol><li>显微镜连接到数字凸轮时代。首先将部分在连接到显微镜扫描的阶段，然后切换到10倍的目标。移动台到感兴趣的区域。 </li><li>启动视频捕捉程序。 </li><li>按下录制按钮。只要记录按钮被按下时，使用宏对焦旋钮从顶部移动到段的底部，总共4秒。保存生成的视频文件。 </li><li>使用ImageJ的免费软件来打开文件并将其保存在非压缩文件格式的AVI视频文件转换成压缩格式。 </li><li>开始图像分析程序并打开该AVI文件。进入"进程"菜单中，单击"扩展景深"。选择相应的视频文件。在"输出"选项中，选择"生成复合的最佳聚焦图像"。 </li><li>在"焦点分析"选项中，选择"归照明"和"最大局部对比度"选项。然后，选择"酶Cre吃了"。所产生的图像代表所有的聚焦像素在整个Z轴（ 图2）。 </li><li>保存生成的图像。 </li></ol> 5.生成极其高清晰度图片注:此方法允许用户自动生成低倍率和高倍率高分辨率图像非常高分辨率的图像在一个自动化的方式。 <ol><li>第一放置部分上连接到所述显微镜的扫描阶段。在显微镜被连接到一台数码相机。移动台到感兴趣的区域。 </li><li>启动图像采集程序。 </li><li>使用10X物镜和取得并保存的高分辨率（4076 X 3116）图片来自感兴趣的区域并确保至少有10％的重叠。 </li><li>运行图像编辑复合缝合图像。 </li><li>转到文件菜单，然后单击"新建全景"。选择其中的图像是ST的文件夹或运算。选择属于同一区域，然后按"确定"按钮图像。 </li><li>按"导出到磁盘"按钮，保存图像。此图像表示感兴趣，可以用来分析和/或演示（ 图3）的区域的超高分辨率图像。 </li></ol>

<ol>
	<li>Bosch, M., Hayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+plasticity+of+dendritic+spines.">Structural plasticity of dendritic spines.</a> Curr Opin Neurobiol. 22 (3), 383-388 (2012).</li><li>Isaac, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+synapse:+center+stage+for+many+brain+diseases.">The synapse: center stage for many brain diseases.</a> The Journal of Physiology. 587 (4), 727-729 (2009).</li><li>Sheng, M., Sabatini, B. L., S&#252;dhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synapses+and+Alzheimer&#8217;s+disease.">Synapses and Alzheimer&#8217;s disease.</a> Cold Spring Harbor Perspectives in Biology. , a005777 (2012).</li><li>Alvarez, V. A., Sabatini, 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=Anatomical+and+physiological+plasticity+of+dendritic+spines.">Anatomical and physiological plasticity of dendritic spines.</a> Annu Rev Neurosci. 30, 79-97 (2007).</li><li>Huttenlocher, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+development+in+neocortex+of+children+with+mental+defect+and+infantile+spasms.">Dendritic development in neocortex of children with mental defect and infantile spasms.</a> Neurology. 24 (3), 203-210 (1974).</li><li>Marin-Padilla, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double+origin+of+the+pericellular+baskets+of+the+pyramidal+cells+of+the+human+motor+cortex:+a+Golgi+study.">Double origin of the pericellular baskets of the pyramidal cells of the human motor cortex: a Golgi study.</a> Brain Res. 38 (1), 1-12 (1972).</li><li>Dang, V., 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=Formoterol,+a+long-acting+&#946;2+adrenergic+agonist,+improves+cognitive+function+and+promotes+dendritic+complexity+in+a+mouse+model+of+Down+syndrome.">Formoterol, a long-acting &#946;2 adrenergic agonist, improves cognitive function and promotes dendritic complexity in a mouse model of Down syndrome.</a> Biol Psychiatry. 75 (3), 179-188 (2014).</li><li>Dobrovi&#263;, B., Curi&#263;, G., Petanjek, Z., Heffer, M. <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+and+spine+density+is+not+altered+in+motor+cortex+and+dentate+granular+cells+in+mice+lacking+the+ganglioside+biosynthetic+gene+B4galnt1+A+quantitative+Golgi+cox+study.">Dendritic morphology and spine density is not altered in motor cortex and dentate granular cells in mice lacking the ganglioside biosynthetic gene B4galnt1 A quantitative Golgi cox study.</a> Coll Antropol. 35 (Suppl 1), 25-30 (2011).</li><li>Dijkmans, T. F., van Hooijdonk, L. W., Fitzsimons, C. P., Vreugdenhil, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+doublecortin+gene+family+and+disorders+of+neuronal+structure.">The doublecortin gene family and disorders of neuronal structure.</a> Cent Nerv Syst Agents Med Chem. 10 (1), 32-46 (2010).</li><li>Guerra, E., Pignatelli, J., Nieto-Est&#233;vez, V., Vicario-Abej&#243;n, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+olfactory+bulb+neurogenesis.">Transcriptional regulation of olfactory bulb neurogenesis.</a> Anat Rec. 296 (9), 1364-1382 (2013).</li><li>Imayoshi, I., Shimojo, H., Sakamoto, M., Ohtsuka, T., Kageyama, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+visualization+of+notch+signaling+in+mammalian+neurogenesis.">Genetic visualization of notch signaling in mammalian neurogenesis.</a> Cell Mol Life Sci. 70 (12), 2045-2057 (2013).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. (65), 3564-3510 (2012).</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>Juraska, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+developmental+plasticity+in+the+visual+cortex+and+hippocampal+dentate+gyrus.">Sex differences in developmental plasticity in the visual cortex and hippocampal dentate gyrus.</a> Prog Brain Res. 61, 205-214 (1984).</li><li>Gao, X., Deng, P., Zao, C. X., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moderate+traumatic+brain+injury+causes+acute+dendritic+and+synaptic+degeneration+in+the+hippocampal+dentate+gyrus.">Moderate traumatic brain injury causes acute dendritic and synaptic degeneration in the hippocampal dentate gyrus.</a> PLoS One. 6 (9), e24566 (2011).</li><li>Zhang, L., Hern&#225;ndez, V. S., Estrada, F. S., Luj&#225;n, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+CA+field+neurogenesis+after+pilocarpine+insult:+The+hippocampal+fissure+as+a+neurogenic+niche.">Hippocampal CA field neurogenesis after pilocarpine insult: The hippocampal fissure as a neurogenic niche.</a> J Chem Neuroanat. 56, 45-57 (2014).</li><li>Merz, K., Lie, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+that+Doublecortin+is+dispensable+for+the+development+of+adult+born+neurons+in+mice.">Evidence that Doublecortin is dispensable for the development of adult born neurons in mice.</a> PLoS One. 8 (5), e62693 (2013).</li><li>Hussaini, S. M., 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=Heat-induced+antigen+retrieval:+an+effective+method+to+detect+and+identify+progenitor+cell+types+during+adult+hippocampal+neurogenesis.">Heat-induced antigen retrieval: an effective method to detect and identify progenitor cell types during adult hippocampal neurogenesis.</a> J Vis Exp. (78), (2013).</li></ol>

这篇文章发表费由医疗保险基金生命科学赞助。

这里，两个方法被描述以量化在使用中与RTI和EDFI结合常规染色方法成熟和新生神经元树突分支的程度。采集的神经元的高分辨率图像提供了一个非常有效的方法，用于测试的神经变性疾病的有害影响，并反过来，提供了一种方法来评估靶向海马神经元的治疗策略。 而RTI方法用于捕获深度数据有关树枝状和地形的程度，所述EDFI方法不能提供有关该个体的段或树木的复杂度信息...

动态改变在突触的数量和结构的发展，老化的标志，以及众多的神经变性疾病1-3。神经元的接收和整合突触信息的能力取决于树枝状形态和动态的改变在突触连接。事实上，一个正相关树突棘和突触数目，这既影响认知功能4之间存在。因此，这并不奇怪，在树突棘数目递减已与认知功能障碍相关的一些神经障碍5-7，促使在树突棘量化了极大的兴趣。然而，脊柱密度的量化仍然是一个耗时且繁琐的任务失败，以产生与通过该树突树突触的地形和分布的有用信息。幸运的是，染色方法（ 如高尔基-考克斯和doublecortin（DCX））结合与先进的成像技术可以被用来克服目前的障碍，并以可靠和迅速的方式产生树突分支的高清晰度图像。而高尔基-考克斯染色法可以部署到评估所有神经元树突8树枝状的状态，DCX可以部署鉴于神经发生在这两个标签新生神经元特别是在齿状回和脑室下区9，一个重要的考虑因素这些地区的整个生命周期10,11。 染色后，两个成像方法被部署，以评估树突特性:ⅰ）实时成像（RTI）和场成像（EDFI）的ⅱ）延伸的深度。的RTI技术提供的平均来跟踪和量化树枝状的长度和顺序沿个体树突段和分支。因此，它使一个估算的总面积和每个树突树占据的体积。多个Specifically，在该RTI方法的用户连续地识别段和迭代地重新聚焦为神经元的跟踪软件收集在x，y和z的树枝状结构的坐标和重构树枝状结构在三维的轨迹。相比之下，EDFI方法提供了一种相当简单的，快速的用于通过产生合成图像，从而提供对整个z轴信息评估相当厚的组织标本树突密度的手段。这样做时，用户记录的高清晰度视频文件贯穿部的厚度，然后使用软件来搜索视频帧以识别点，其中一个像素是完全聚焦。随后，将聚焦像素被合并和集成到一个高分辨率，复合2D图像。这种复合图象包含分别在焦，不论其在z轴位置的所有像素。这些2D图像的定性和定量分析随后可以用来确定密度的树枝状分枝中的每个字段。 最后，我们提出了一个全景方法用于产生极高分辨率的图像进行分析和树突的评估在感兴趣的整个区域。这种技术可以被部署到克服缺乏接入的到非常高的分辨率和昂贵的数码相机。使用这种方法，因此一个捕获在沿x轴和y轴的不同位置的序列的图像，然后自动缝合在一起使用免费软件（ 例如 ，图片复合编者）。值得注意的是，可以在一个相当宽的区域被用于树突分支的定性和定量评估此方法。

<table><tbody><tr><td>Modified Golgi-cox staining solution&amp;nbsp;</td><td>Weill Cornell Medical College</td><td>NA</td><td>store at 4&amp;deg;C till use</td></tr><tr><td>1x Developing Solution (Stock 10x)</td><td>Weill Cornell Medical College</td><td>NA</td><td>store at 4&amp;deg;C till use</td></tr><tr><td>30% Sucrose,</td><td>Sigma</td><td>CAS # 57-50-1</td><td>make fresh &amp;nbsp;in ddH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>0.3% Gelatin</td><td>Sigma</td><td>CAS # 9000-70-8</td><td>NA</td></tr><tr><td>Graded Ethanol Solutions (20%, 30%, 40%, 50%, 80%. 90%, 95%. 100%)</td><td>Sigma</td><td>CAS 603-003-00-5</td><td>NA</td></tr><tr><td>Xylene</td><td>Sigma</td><td>CAS # 1330-20-7</td><td>NA</td></tr><tr><td>DPX Medium</td><td>EMS&amp;nbsp;</td><td>#13510</td><td>NA</td></tr><tr><td>Superfrost (+) white</td><td>Electron Microscopy Sciences</td><td>71869-10</td><td>NA</td></tr><tr><td>Coverslip 22x50mm (VWR #48393-059)</td><td>VWR&amp;nbsp;</td><td>#4811-703</td><td>NA</td></tr><tr><td>DCX Antibody</td><td>Santa Cruz Biotechnology</td><td>sc-8066</td><td>4 C</td></tr><tr><td>DAB</td><td>Sigma</td><td>CAS Number 91-95-2 &amp;nbsp;</td><td>-20</td></tr><tr><td>OCT</td><td>Tissue-tek</td><td>4583</td><td>NA</td></tr><tr><td>Tris</td><td>Sigma</td><td>CAS Number 77-86-1 &amp;nbsp;</td><td>NA</td></tr><tr><td>ABC Lite</td><td>Vector</td><td>PK4000</td><td>NA</td></tr><tr><td>Microscope</td><td>Nikon</td><td>Eclipse 80i</td><td></td></tr><tr><td>Digital Camera</td><td>Nikon</td><td>DS-Ri1</td><td></td></tr><tr><td>12 bit Camera&amp;nbsp;</td><td>QImaging&amp;nbsp;</td><td>01 MBF2000RF-CLR-12</td><td></td></tr><tr><td>Neurolucida System</td><td>MBF Bioscience</td><td>V.10</td><td></td></tr><tr><td>Image Composite Editor</td><td>Microsoft</td><td>1.4.4.0</td><td></td></tr><tr><td>NIS Elements</td><td>Nikon</td><td>F 3.0</td><td></td></tr><tr><td>Image Pro Plus</td><td>Mediacy</td><td>Versin 7.00</td><td></td></tr></tbody></table>

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.

从现存的和刚出生的齿状颗粒细胞产生树枝状的程度用两种高尔基-考克斯或DCX染色（ 图1）在野生型小鼠进行分析。 DCX阳性细胞的树突状段被发现是13-36微米长。使用Kolmogorov-Smirnov检验树突长度的正态分布进行了测试（D = 0.1217，P &lt;0.01，Liliefors P &lt;0.001; 图4和5）。 在每个订单树枝状的长度段的分析，最长的片段被发现在树枝状（38.38 + ...

Watch this Scientific Journal Video about Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice at JoVE.com

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

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 ...

assessment-dendritic-arborization-dentate-gyrus-hippocampal-region

Research

JoVE Journal

Neuroscience

10.2K 次观看.  VA Palo Alto Health Care System. 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.

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

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

科研

行为学

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

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

生物化学

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 ...

神经科学

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

海马鼠脑切片的突触密度评价

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

An Invasive Method for the Activation of the Mouse Dentate Gyrus by High-frequency Stimulation

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for various neurological and psychiatric disorders. HFS in the deep region of the brain, also named deep-brain stimulation (DBS), is becoming increasingly important in clinical trials. Recent progress in the field of high-frequency DBS (HF-DBS) surgery has begun to spread the possibility of utilizing this invasive technique to other situations, such as treatment for major depression disorder (MDD), obsessive-compulsive disorder (OCD), and so on. 
Despite these expanding indications, the underlying mechanisms of the beneficial effects of HF-DBS remain enigmatic. To address this question, one approach is to use implanted electrodes that sparsely activate distributed subpopulations of neurons by HFS. It has been reported that HFS in the anterior nucleus of the thalamus could be used for the treatment of refractory epilepsy in the clinic. The underlying mechanisms might be related to the increased neurogenesis and altered neuronal activity. Therefore, we are interested in exploring the physiological alterations by the detection of neuronal activity as well as neurogenesis in the mouse dentate gyrus (DG) before and after HFS treatment. 
In this manuscript, we describe methodologies for HFS to target the activation of the DG in mice, directly or indirectly and in an acute or chronic manner. In addition, we describe a detailed protocol for the preparation of brain slices for c-fos and Notch1 immunofluorescent staining to monitor the neuronal activity and signaling activation and for bromodeoxyuridine (BrdU) labeling to determine the neurogenesis after the HF-DBS induction. The activation of the neuronal activity and neurogenesis after the HF-DBS treatment provides direct neurobiological evidence and potential therapeutic benefits. Particularly, this methodology can be modified and applied to target other interested brain regions such as the basal ganglia and subthalamic regions for specific brain disorders in the clinic.

This protocol shows how to set up a reliable HFS method in mice. Neurons throughout the hippocampal dentate gyrus are electrically stimulated by HFS directly and indirectly in vivo. Neuronal activity and molecular signaling are examined by c-fos and Notch1 immunofluorescent staining, respectively; neurogenesis is quantified by bromodeoxyuridine labeling assay.

高频刺激激活小鼠齿状回的侵袭方法

Electrical high-frequency stimulation (HFS), using implanted electrodes targeting various brain regions, has been proven as an effective treatment for ...

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 ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

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.

树突分支中的海马区对小鼠的齿状回评

摘要

探索更多视频

此视频中的章节