Name: use of synaptic zinc histochemistry to reveal different regions and laminae in the developing and adult brain
Uploaded: 2017-10-29T13:00:00.000+00:00
Duration: 9 min 50 s
Description: Watch this Scientific Journal Video about Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain at JoVE.com

这项工作得到了国家研究资源中心 (2G12RR03060-26A1) 的赠款的支持;国立卫生研究院的少数群体健康和健康差异 (8G12MD007603-27);纽约市立大学专业工作人员代表大会和教职员研究津贴 (悬浮角速度陀螺仪 II) 美国沙迦大学。我们感谢维德雅瑟格 Sriramoju 向我们介绍这些方法。

以下协议遵循由纽约市城市学院机构动物保育和使用委员会 (IACUC) 建立的动物保育指导方针, 该准则符合所有适当的州和联邦准则。麻醉适合于雪貂, 并应根据所研究的物种进行修改. 
 <img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56547/56547fig1.jpg"/> 图 1: 流程图概述了本协议3阶段涉及的主要步骤以及完成每个步骤所需的时间. 要求部分完全干燥的期间显示在绿色文本圆圈中, 而所有其他步骤都在白色文本圆圈中。绿色菱形文本框是一个决策点, 而红色矩形是一个关键步骤, 应该格外小心地执行。<a href="//ecsource.jove.com/files/ftp_upload/56547/56547fig1large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
 1. 准备步骤 (幻灯片单调和解决方案) <ol> <li> 在热水中用洗涤剂清洗 unsubbed 的幻灯片, 然后在温水中冲洗几次, 然后用蒸馏水冲洗, 彻底清除污垢或碎屑。允许幻灯片在室温或 37 #176 的烤箱中干燥; C. </li>
	<li> 一旦幻灯片完全干燥, 每张幻灯片上都用手指或画笔将一层薄薄的蛋清均匀地涂上。允许干燥在烤箱在60和 #176; C 为 20-30 min。为了获得最佳效果, 并避免部分滑脱, 添加第二层蛋清, 并允许在60和 #176 的烤箱再干一次; C. </li>
	<li> 在100毫升和 #160 中溶解1克的明胶, 在热水 (60 和 #176; C) 中制备1% 明胶溶液, 使其冷却至室温. </li>
	<li> 准备 200 mL 开发人员解决方案, 如下所述, 供4节使用. 
	<ol> <li> 通过缓慢地将40克加到120毫升的热水中来准备阿拉伯树胶解决方案 (这种方法更容易溶解)。继续搅拌的解决方案与玻璃搅拌棒。一旦溶液完全溶解, 将其从热量中取出, 让它冷却几分钟, 然后在漏斗中通过 6-8 层纱布滤布. </li>
		<li> 在20毫升 dH 中加入5.04 克柠檬酸加4.7 克柠檬酸钠, 配制柠檬酸缓冲液 2 O 溶解混合物。确保本解决方案的 ph 值为 4.0, 在25和 #176; c. 在必要时通过在溶液中加入氢氧化钠或盐酸来调节 ph 值. </li>
		<li> 通过加热30毫升 dH 2 O 并溶解1.7 克氢醌来制备氢醌溶液. 
		注: 加热氢醌溶液对于使其在水中容易溶解很重要, 因为氢醌在室温下不易溶解于水中。注意保持水温低于 60 #176; C, 否则氢醌可能被氧化。如果解决方案变成黄色, 丢弃并准备另一个新的解决方案. </li>
		<li> 在30毫升的 dH 中溶解0.22 克, 制备银乳酸溶液 2 O. </li>
		<li> 将解决方案 (1.4.1-1.4.4) 按它们在准备执行反应时所描述的顺序 (即, 切片、干燥和固定后) ( 图 2 ) 混合。最后加入乳酸银溶液。确保此步骤很快完成, 开发人员解决方案被放置在黑暗中, 直到它是反应的部分, 因为银乳酸是光敏. </li>
	</ol> </li> </ol> <img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56547/56547fig2.jpg"/> 图 2: 示意图中涉及的步骤序列在该协议的锌组织化学阶段混合试剂. <a href="//ecsource.jove.com/files/ftp_upload/56547/56547fig2large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
 2. 动物治疗和麻醉 <ol> <li> 在镇静动物之前, 准备1% 亚硒酸钠 (Na 2 SeO 3 ) 解决方案, 在10毫升 dH 1 O 中溶解2毫克的亚硒酸钠。
	<li> 麻醉肌肉注射氯胺酮 (25 毫克/千克) 和嗪 (2 毫克/千克) 的动物. 
	注意: 通过使用踏板反射反应, 确保达到适当的麻醉水平. </li>
	<li> 注入锌合剂亚硒酸钠溶液 (15 毫克/千克) 腹腔 (IP). 
	注意: 亚硒酸钠在不同年龄或其他种类的毒性可能有所不同. </li>
	<li> 允许60至90分钟的亚硒酸钠前往大脑。在此期间, 必须确保该动物是适当的镇静剂和没有反应, 所以检查深度的麻醉每5分钟. </li>
	<li> 在亚硒酸盐期间, 当动物被麻醉时, 确保眼睛关闭以防止干燥, 或管理眼部药膏以保持湿润. </li>
</ol> 3。组织准备和染色 <ol> <li> 通过管理戊巴比妥钠过量 (100 毫克/千克, i. p) 来安乐该动物. </li>
	<li> 用生理盐水进行 transcardial 灌注1分钟, 并与4% 甲醛20分钟, 最后, 管理一个解决方案与4% 甲醛和10% 蔗糖 (总固定期 1 h). </li>
	<li> 使用一对大剪刀移除磁头. </li>
	<li> 用手术刀从鼻子到颈部进行中线切口, 以露出颅骨. </li>
	<li> 小心地取出大脑, 用刀片将半球分开. </li>
	<li> 在0.1 米磷酸缓冲液 (PB) 中阻断大脑后部和4% 甲醛的后缀数小时. </li>
	<li> 将块放入30% 的蔗糖溶液中, 在0.1 米 PB 中, 并允许大脑下沉. 
	注: 在0.1 米 PB 中, 用30% 蔗糖溶液取代4% 甲醛和30% 蔗糖溶液, 使大脑的大脑和 #39; 甲醛的暴露, 这会影响组织染色质量. </li>
	<li> 一旦大脑下沉, 切割 semi-tangential 40 和 #181; m 厚部分通过视觉皮层或区域的兴趣在冻结, 滑动切片或低温。这可以通过放置块与内侧表面下来, 并轻轻地拼合玻璃幻灯片. </li>
	<li> 用画笔收集剖面, 并将其存储在含有磷酸盐缓冲盐水 (PBS) 的钓具盒中. </li>
	<li> 将各节分隔为单独的编号系列。立即登上一个或两个系列的大脑部分的卵白色眼见幻灯片 (1 节), 允许干燥过夜在室温下, 和工艺部分 histochemically 突触锌. 
	注: 其他系列可以处理其他标记进行比较, 如髓鞘 12 或细胞色素氧化酶使用修改后的协议 13 : 孵育 2-8 h 在40和 #176; c 与3% 蔗糖, 0.015% 细胞色素 c,0.015% 过氧化氢酶, 0.02% diaminobenzidine 0.1 米 PB。尼物质也可被用作鉴别视觉皮层区域的组织学标记。这些其他组织学污点不要求大脑部分安装在蛋清眼见片上, 因此可以改用传统的明胶涂层的幻灯片. </li>
</ol> 4。突触锌组织化学 <ol> <li> 在绝对酒精中固定滑动段15分钟, 并允许在室温下完全干燥1小时. </li>
	我我简要地沉浸在十年代的1% 明胶溶液 (第1节), 并允许在室温下过夜干燥. 
	注意: 为了获得最佳效果, 允许部分在一夜之间干燥。允许部分干燥过夜会产生更好的组织染色. </li>
	<li> 将这些解决方案混合在一起, 如步骤1.8 中所述, 一旦部分准备就绪, 将立即做出反应. 
	<ol> <li> 通过在玻璃或塑料托盘中并排排列幻灯片, 并将开发的解决方案倒在幻灯片上, 来响应各节。验证幻灯片是否完全淹没在解决方案中, 并将纸盒传输到暗空间, 或用光框覆盖. 
		注: 大约12英寸长8英寸宽的塑料托盘可以使用, 正好适合18张幻灯片。200 mL 开发人员解决方案的总容量足以完全淹没幻灯片, 因此请确保正确的音量用于幻灯片的数量, 并相应地调整食谱。使用塑料或玻璃托盘, 而不是使用金属托盘是可取的, 因为有一定程度的交叉反应之间的银乳酸的开发商解决方案和铁或其他金属发现金属托盘. </li>
	</ol> </li> <li> 通过直观地检查每30分钟的节来监视该反应的发展。这些部分一般需要 120-180 分钟的完整开发. </li>
	<li> 如果部分变为 overstained (请参见 图 3a ), 请在2% 农民和 #39; s 解决方案 (dh 中9份2% 硫代硫酸钠 2 o 和1部分2% 钾氰化钾在 dh 2 o) 中 1-2 min. 
	注意: 未着色的样本部分显示在 图 3b 中. </li>
	<li> 达到所需强度后, 通过从纸盒中卸下幻灯片安装的部分并将幻灯片放在幻灯片机架上来终止反应. </li>
	<li> 将滑轨架放在一个大的玻璃染色盘中, 然后在温水 (40-50 和 #176; C) 中冲洗幻灯片10分钟以除去明胶涂层和外银沉积物. 
	注意: 小心不要搅动滑动架以防止部分滑落。当充分的层流变化是明显的, 并且区段是足够黑暗的, 但没有反应过度 (参见 图 4a ) 时, 期望的部分的强度达到. </li>
	<li> 允许幻灯片在室温下干燥, 然后在100% 乙醇 (5 分钟) 内脱水, 在二甲苯 (5 分钟) 内清除, 并以安装介质覆盖滑块。或者, 将幻灯片放在上升的酒精系列中, 然后脱水、清除和片. </li>
	<li> 使用不含亚硒酸前处理的动物的切片作为阴性对照。这些部分的银放大不应产生染色. </li>
</ol> <img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56547/56547fig3v2.jpg"/> 图 3: 幼鼬脑中的突触锌染色. 显微 semi-tangential overstained 和 b) understained 在幼年的雪貂脑中。由于缺乏层流变化, 区域边界难以辨别。白色物质也被严重污染。Ssy Suprasylvian 皮质, WM 白质, 前, D 背。缩放条 = 500 和 #181; m (a-b)。<a href="//ecsource.jove.com/files/ftp_upload/56547/56547fig3v2large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
 5. 区分区域边界和图像获取 <ol> <li> 使用不同脑区的建筑特征进行平面和层流识别. 
	注: 例如, 在发育中的大鼠压皮层 14 中, 作者揭示了一种瞬态模块化的特点是锌的重染色, 这是不存在于成人, 但可以用来描述皮质在这个物种的发展过程中的组织。在另一项研究 15 中, 作者揭示了猕猴杏仁核中发现的不同核的突触锌分布的特异性, 这有助于鉴别这些分裂。在发育和成人鼬脑的视觉皮层区域以前被描述了 16 , 17 , 18 , 建筑中大脑皮层的细分灰松鼠被描述了 19 。此外, 锌的组织化学以前被用来区分成年猴视觉皮层的区域 8 , 发育和成年猫视觉皮层 5 , 发育中的大鼠体感皮层 9 , 20 和成年老鼠体感皮层 6 , 21 。如果可用, 比较髓磷脂染色, 细胞色素氧化酶 (CO) 染色, 和突触锌染脑切片, 以确认在成人的区域边界。在 semi-tangential 部分的雪貂视觉皮层染色的突触锌, 有明显的差异, 在视觉皮层区, 以促进区域识别。例如, 17 区和18的成人鼬揭示, 突触锌染色是高的层 I III 和 v 层的污渍不太强烈, 而第四层几乎缺乏锌。明显缺乏锌染色在 iv 层或区域17和18对比与黑暗地被发现的色带在 CO 被染色的部分。然而, 在 CO 染色的区域中, 17 的第四层保持了与 iii 和 V 层的尖锐边界, 但18区的第四层的特征是染色强度有细微的下降, 第三层中发现的上边界较不易区分. </li>
	<li> 使用低功耗目标 (2X 或4X 放大倍数) 的明显微镜检查剖面, 并拍摄感兴趣的区域. </li>
	<li> 使用图像处理软件增强显微的对比度和亮度。光学密度测量所获得的图像不应以任何方式改变. </li>
</ol> <img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56547/56547fig4v3.jpg"/> 图 4: 成人雪貂脑中的突触锌染色区别不同的视觉皮层区域. 显微相邻的 semi-tangential 部分染色为 ( a ) 突触锌或 ( b ) 细胞色素氧化酶 (CO) 在成年。箭头标记区域边界。Ssy Suprasylvian 皮层, 前, D 背。标尺 = 500 和 #181; m. <a href="//ecsource.jove.com/files/ftp_upload/56547/56547fig4v2large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a> 
 6. 密度测定 (可选) 注意: Densitometric 分析可用于评估在感兴趣的区域内有代表性的锌染色切片的光学密度, 从而评价突触锌在大脑中的分布。这种方法也有助于跟踪整个开发过程中突触锌水平的潜在变化. 
<ol> <li> 使用选定的锌染色切片, 随机选择适当宽度的皮质柱 (柱状显微) (450 和 #181; m 宽柱) 从所获得的感兴趣区域的显微。皮质柱是横跨所有皮层层从脑膜表面到白质的区域. </li>
	<li> 从每个感兴趣区域的几个不同的大脑部分中选择适当数量的样本列. </li>
	<li> 将具有代表性的列的示例图像传输到图像处理软件中. </li>
	<li> 使用矩形选择工具来包含整个皮质列. </li>
	<li> 使用反转工具创建与该列的负片类似的对比度反转图像. 
	注: 对高突触锌水平和低突触锌含量低的光学密度值, 图像进行对比反演, 以产生高的光学密度值。这是一种更直观的方式来呈现类似于 图 5 中所示的图形轮廓图. </li>
	<li> 通过使用绘图剖面工具生成这些图像中的光学密度剖面图, 以便沿直线产生像素强度的 two-dimensional 图形. </li>
	<li> 使用绘图选项将绘图剖面图转换为垂直配置文件, 然后再次单击 "绘图" 配置文件. 
	注意: x 轴表示沿直线的距离, y-axis 表示像素强度。因此, 每个图块配置文件值都反映了跨列宽的每个深度的平均灰度值. </li>
	<li> 将绘图配置文件值作为电子表格中的文本文件打开, 将其规范化并绘制为图形 (请参见 图 5 ). 
	注意: 最好使用突触锌的相对密度作为定量测量的比较结果, 由于不同的反应时间、组织的 stainability 以及其他变量. </li>
	<li> 通过首先执行车厢平均的绘图剖面值来计算相对锌密度, 以平滑数据. 
	注意: 这是通过平均, 例如, 每连续20或30像素 (1 像素 = 2.5 和 #181; m) 的深度, 然后正常化, 以最大强度为每个样本。因此, 每个平均绘图剖面值反映的是该深度的平均灰度值 (灰度值范围从0到 255)。一种不同的归一化方法可用于首先获取样本区域的白质 (WM) 光学密度值, 其中包括感兴趣区域中的底层白质。理想情况下, 选择几个区域, 尽可能轻的染色, 获得一个平均 WM 值。平均光学密度值然后除以平均值 wm 值, 以获得 wm 正常化值. </li>
	<li> 确定在感兴趣区域的特定层中的平均光学密度值以进行定量比较. 
	注: 例如, 雪貂视觉皮层区第四层的平均最小光学密度值是由包含最小染色区域的 #177; 5 像素. </li>
	<li> 在雪貂的视觉皮层区域的 supragranular 和 infragranular 层中计算平均光学密度值, 由 #177 的最暗的区域组成; 5 像素以确定平均值的最大值. </li>
	<li> 确保平均光学密度值是从特定的层中获得的. 
	注意: 在对比反转图像中, 通过将它们与原显微以及相邻的 CO 剖面进行比较, 验证这些层的极限是非常必要的。这保证了一个不会侵入相邻的层. </li>
</ol> <img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56547/56547fig5.jpg"/> 图 5: 不同视觉皮层突触锌的层流分布区域在成人雪貂. 有代表性的显微的柱子通过所有皮层层与相应的规范化的光学密度剖面在成人。低突触锌密度在成人地区 IV 层17和18是由槽在剖面图中表示。在每个绘图剖面中, IV 层槽中的填充椭圆表示用于确定平均最小像素强度值的值。标尺 = 200 和 #181; m. <a href="//ecsource.jove.com/files/ftp_upload/56547/56547fig5large.jpg" target="_blank"> 请单击此处查看此图的较大版本. </a>

<ol>
	<li>Nakashima, A., Dyck, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+and+cortical+plasticity.">Zinc and cortical plasticity.</a> Brain Res. Rev. 59, 347-373 (2009).</li><li>Frederickson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+zinc+and+zinc-containing+neurons.">Neurobiology of zinc and zinc-containing neurons.</a> Int Rev Neurobiol. 31, 145-238 (1989).</li><li>Beaulieu, C., Dyck, R., Cynader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+glutamate+in+zinc-containing+terminals+of+the+cat+visual+cortex.">Enrichment of glutamate in zinc-containing terminals of the cat visual cortex.</a> NeuroReport. 3 (10), 861-864 (1992).</li><li>Martinez-Guijarro, F. J., Soriano, E., Del Rio, J. A., Lopez-Garcia, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc-positive+boutons+in+the+cerebral+cortex+of+lizards+show+glutamate+immunoreactivity.">Zinc-positive boutons in the cerebral cortex of lizards show glutamate immunoreactivity.</a> J Neurocytol. 20 (10), 834-843 (1991).</li><li>Dyck, R., Beaulieu, C., Cynader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+localization+of+synaptic+zinc+in+the+developing+cat+visual+cortex.">Histochemical localization of synaptic zinc in the developing cat visual cortex.</a> J Comp Neurol. 329 (1), 53-67 (1993).</li><li>Garrett, B., Geneser, F. A., Slomianka, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+acetylcholinesterase+and+zinc+in+the+visual+cortex+of+the+mouse.">Distribution of acetylcholinesterase and zinc in the visual cortex of the mouse.</a> Anat Embryol. (Berl). 184 (5), 461-468 (1991).</li><li>Garrett, B., Osterballe, R., Slomianka, L., Geneser, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoarchitecture+and+staining+for+acetylcholinesterase+and+zinc+in+the+visual+cortex+of+the+Parma+wallaby+(Macropus+parma).">Cytoarchitecture and staining for acetylcholinesterase and zinc in the visual cortex of the Parma wallaby (Macropus parma).</a> Brain Behav Evol. 43 (3), 162-172 (1994).</li><li>Dyck, R., Cynader, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+interdigitated+columnar+mosaic+of+cytochrome+oxidase,+zinc,+and+neurotransmitter-related+molecules+in+cat+and+monkey+visual+cortex.">An interdigitated columnar mosaic of cytochrome oxidase, zinc, and neurotransmitter-related molecules in cat and monkey visual cortex.</a> Proc. Natl. Acad. Sci. (90), 9066-9069 (1993).</li><li>Land, P. W., Akhtar, N. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+alteration+of+synaptic+zinc+in+rat+somatosensory+barrel+cortex.">Experience-dependent alteration of synaptic zinc in rat somatosensory barrel cortex.</a> Somatosens Mot Res. 16 (2), 139-150 (1999).</li><li>Danscher, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exogenous+selenium+in+the+brain:+a+histochemical+technique+for+light+and+electron+microscopic+localization+of+catalytic+selenium+bonds.">Exogenous selenium in the brain: a histochemical technique for light and electron microscopic localization of catalytic selenium bonds.</a> Histochemistry. 76, 281-293 (1982).</li><li>Danscher, G., Howell, G., Perez-Clausell, J., Hertel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dithizone,+Timm's+sulphide+silver+and+the+selenium+methods+demonstrate+a+chelatable+pool+of+zinc+in+CNS:+a+proton+activation+(PIXE)+analysis+of+carbon+tetrachloride+extracts+from+rat+brains+and+spinal+cords+intravitall+treated+with+dithizone.">The dithizone, Timm's sulphide silver and the selenium methods demonstrate a chelatable pool of zinc in CNS: a proton activation (PIXE) analysis of carbon tetrachloride extracts from rat brains and spinal cords intravitall treated with dithizone.</a> Histochemistry. 83, 419-422 (1985).</li><li>Gallyas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+staining+of+myelin+by+means+of+physical+development.">Silver staining of myelin by means of physical development.</a> Neurol Res. 1 (2), 203-209 (1979).</li><li>Wong-Riley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+visual+system+of+monocularly+sutured+or+enucleated+cats+demonstrable+with+cytochrome+oxidase+histochemistry.">Changes in the visual system of monocularly sutured or enucleated cats demonstrable with cytochrome oxidase histochemistry.</a> Brain Res. 171 (1), 11-28 (1979).</li><li>Mir&#243;-Berni&#233;, N., Ichinohe, N., Perez-Clausell, J., Rockland, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc-rich+transient+vertical+modules+in+the+rat+retrosplenial+cortex+during+postnatal+development.">Zinc-rich transient vertical modules in the rat retrosplenial cortex during postnatal development.</a> J Neurosci. 138 (2), 523-535 (2006).</li><li>Ichinohe, N., Rockland, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+synaptic+zinc+in+the+macaque+monkey+amygdala.">Distribution of synaptic zinc in the macaque monkey amygdala.</a> J Comp Neurol. 489 (2), 135-147 (2005).</li><li>Innocenti, G. M., Manger, P. R., Masiello, I., Colin, I., Tettoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+and+callosal+connections+of+visual+areas+17,+18,+19+and+21+in+the+ferret+(Mustela+putorius).">Architecture and callosal connections of visual areas 17, 18, 19 and 21 in the ferret (Mustela putorius).</a> Cereb Cortex. 12 (4), 411-422 (2002).</li><li>Khalil, R., Levitt, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zinc+histochemistry+reveals+circuit+refinement+and+distinguishes+visual+areas+in+the+developing+ferret+cerebral+cortex.">Zinc histochemistry reveals circuit refinement and distinguishes visual areas in the developing ferret cerebral cortex.</a> Brain Struct Funct. 218, 1293-1306 (2013).</li><li>Manger, P. R., Masiello, I., Innocenti, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Areal+organization+of+the+posterior+parietal+cortex+of+the+ferret+(Mustela+putorius).">Areal organization of the posterior parietal cortex of the ferret (Mustela putorius).</a> Cereb Cortex. 12, 1280-1297 (2002).</li><li>Wong, P., Kaas, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architectonic+subdivisions+of+neocortex+in+the+gray+squirrel+(Sciurus+carolinensis.).">Architectonic subdivisions of neocortex in the gray squirrel (Sciurus carolinensis.).</a> The anatomical record. 291, 1301-1333 (2008).</li><li>Land, P. W., Shamalla-Hannah, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experience-dependent+plasticity+of+zinc-containing+cortical+circuits+during+a+critical+period+of+postnatal+development.">Experience-dependent plasticity of zinc-containing cortical circuits during a critical period of postnatal development.</a> J Comp Neurol. 447 (1), 43-56 (2002).</li><li>Czupryn, A., Skangiel-Kramska, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+synaptic+zinc+in+the+developing+mouse+somatosensory+barrel+cortex.">Distribution of synaptic zinc in the developing mouse somatosensory barrel cortex.</a> J Comp Neurol. 386, 652-660 (1997).</li><li>Timm, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zur+Histochemie+der+Schwermetalle.+Das+Sulfid-Silber-Verfahren.">Zur Histochemie der Schwermetalle. Das Sulfid-Silber-Verfahren.</a> Dtsch Z ges gerichtl Med. 46, 706-711 (1958).</li></ol>

作者没有什么可透露的。

目前的研究采用了基于改进版本的 Danscher (1982) 方法10的组织化学技术, 据此可以在大脑中检测到突触锌的定位并进行可视化。这种方法基本上是通过注射合剂酸钠 (Na2SeO3) (15 毫克/千克) 的动物。在注射后, 亚硒酸向脑部移动, 并与游离锌结合, 前囊泡中含有锌的神经元。锌离子与突触囊泡内的分子结合沉淀, 并可通过物理发育2,<sup class="xre...

组织学上的污点传统上被用来帮助鉴别不同物种的皮质区, 揭示不同的建筑特征。联合使用的组织化学技术, 如尼物质, 细胞色素氧化酶 (CO) 的反应性, 或髓鞘可以证明是卓有成效的, 因为他们揭示了类似的区域边界在成人的大脑。然而, 这些组织化学染色并不总能充分揭示未成熟大脑皮层区和层间的明显界限。
在中枢神经系统, 锌有几个关键的功能, 包括稳定 DNA 结构, 作为一个酶因子, 参与许多调节功能, 并作为一个调通过其存在的突触囊泡1. 突触锌是独一无二的, 因为它可以使用组织学方法进行可视化, 而蛋白质结合的锌则不能被形象化2。该功能已被用来揭示不同皮层区的突触锌模式, 并在许多研究中使用突触锌组织化学。大脑皮层中谷氨酸神经元的一个子集在其轴突末端的前泡内含有锌,3,4。组织化学研究揭示了脑皮层突触锌的异质分布5,6,7。在不同皮层区 (如, 视觉与体感皮层) 或层 (如如, 锌含量在 supragranular 和infragranular 层的初级视觉皮层大大高于丘脑输入层 IV 与相对较低的突触锌水平)5,8,9。在皮层中观察到的突触锌染色的异质性特别有利, 因为它能促进区域和层流的识别。
在这里, 我们提出了一个突触锌组织化学过程的详细描述, 这是一个修改版本的 Danscher 的1982方法10。该方法利用亚硒酸腹腔 (IP) 作为螯合剂。亚硒酸的移动到大脑, 以反应池的自由锌发现在囊泡的一个子集的谷氨酸突触在大脑中。这种反应产生的沉淀, 可以提高随后由银开发2,10,11。
这一过程揭示了突触锌染色的层流和区域模式;密度分析可用于评估这些模式的质量和数量在成人和未成熟的大脑, 以研究影响的其他干预, 如感官, 环境, 药理, 或基因的操纵。此外, 人们还可能希望评估其他模型系统中其他皮层或皮层下结构中的突触锌分布的潜在发展变化。密度分析提供的定量信息对后续的脑发育具有一定的优势。该协议提供了一个同伴的其他免疫和组织化学标记, 以揭示层流和区域边界。

<table><tbody><tr><td>Euthasol (Euthanasia solution)</td><td>Henry Schein</td><td>710101</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma-Aldrich</td><td>214485</td><td></td></tr><tr><td>Ketamine (Ketaved)</td><td>Henry Schein</td><td>48858</td><td>100 mg/ml injectables</td></tr><tr><td>Xylazine (Anased)</td><td>Henry Schein</td><td>33198</td><td>100 mg/ml injectables</td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>F8775</td><td>Dilute to 4%</td></tr><tr><td>Gum arabic</td><td>Sigma-Aldrich</td><td>G9752-500G</td><td></td></tr><tr><td>Citric acid</td><td>Sigma-Aldrich</td><td>C1909</td><td></td></tr><tr><td>Sodium citrate</td><td>Sigma-Aldrich</td><td>W302600</td><td></td></tr><tr><td>Hydroquinone</td><td>Sigma-Aldrich</td><td>H9003</td><td></td></tr><tr><td>Silver lactate</td><td>Sigma-Aldrich</td><td>85210</td><td></td></tr><tr><td>Fish gelatine</td><td>Sigma-Aldrich</td><td>G7765</td><td></td></tr><tr><td>Cytochrome c</td><td>Sigma-Aldrich</td><td>C2506</td><td>(Type III, from equine heart)</td></tr><tr><td>Catalse</td><td>Sigma-Aldrich</td><td>C10</td><td></td></tr><tr><td>Sucrose</td><td>Domino</td><td></td><td></td></tr><tr><td>Xylene</td><td>Fisher Scientific</td><td>X5P-1GAL</td><td></td></tr><tr><td>Permount</td><td>Fisher Scientific</td><td>SP15-500</td><td></td></tr><tr><td>100% Ethanol</td><td>Fisher Scientific</td><td>A406-20</td><td>Used for dehydration prior to slide mounting</td></tr><tr><td>Coverslips</td><td>Brain Research Laboratories</td><td>#3660-1</td><td></td></tr><tr><td>Frosted unsubbed slides</td><td>Brain Research Laboratories</td><td>#3875-FR</td><td></td></tr><tr><td>Microtome</td><td>American Optical Company</td><td>860</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>BX-60</td><td></td></tr><tr><td>Adope Photoshop</td><td>Adobe Systems, San Jose, CA</td><td></td><td>To assemble images</td></tr><tr><td>ImageJ</td><td>Free software can be downloaded at http://rsb.info.nih.gov/ij/</td><td></td><td>For densometric measurements</td></tr><tr><td>Plastic tray</td><td>Any standard plastic tray may be used</td><td></td><td>to immerse slides in developer solution</td></tr><tr><td>Hot plate</td><td>Any standard hotplate may be used</td><td></td><td></td></tr></tbody></table>

use of synaptic zinc histochemistry to reveal different regions and laminae in the developing and adult brain

解剖和功能性脑组织和发展的特征, 需要准确识别不同的神经回路和未成熟和成人大脑的区域。在这里, 我们描述了一个锌组织化学染色程序, 揭示不同的染色模式之间的各层和脑区。其他人利用这一程序不仅揭示了含锌神经元的分布和大脑中的电路, 而且成功地描绘了几个物种的发育中和成年大脑的区域和层流边界。在这里, 我们说明这个染色程序与图像从开发和成人鼬脑。我们揭示了一个锌染色模式, 作为一个解剖标记的地区和层, 并可以可靠地用于区分视觉皮层区域的发展和成人视觉皮层。本议定书的主要目的是提出一种组织化学方法, 允许准确地识别发育中和成人大脑中其他方法无法做到的层和区域。其次, 结合密度图像分析, 这种方法可以评估突触锌的分布, 以揭示整个开发过程中的潜在变化。本协议详细描述了连续染色冰冻脑切片所需的试剂、工具和步骤。虽然这个协议被描述使用鼬脑组织, 它可以很容易地被适应用于啮齿目动物, 猫, 或猴子并且在其他脑子区域。

在图 1的流程图中, 本协议中涉及的主要步骤是为突触锌的大脑切片着色。该协议可分为三阶段: 1) 灌注和组织收集, 2) 组织制备和染色, 3) 锌组化学。简单地, 在协议的第一阶段, 动物被麻醉并且注射了 IP 以适当的剂量亚硒酸钠。在一个充足的时间段 (理想情况下 60-90 min) 之后, 该动物随后被安乐死, 用 0.9% NaCl 溶液灌流, 然后是4% 甲醛溶液。头部被斩首...

Watch this Scientific Journal Video about Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain at JoVE.com

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

利用突触锌组织化学在发育和成人大脑中揭示不同的区域和叶片

我们描述了一个组织化学过程, 揭示特征的层流和区域锌染色模式在不同的脑区。锌染色模式可与其他解剖标记一起使用, 以可靠地区分发育中和成人大脑的层和区域。

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...

use-synaptic-zinc-histochemistry-to-reveal-different-regions-laminae

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

7.1K 次观看.  American University of Sharjah. 我们描述了一个组织化学过程, 揭示特征的层流和区域锌染色模式在不同的脑区。锌染色模式可与其他解剖标记一起使用, 以可靠地区分发育中和成人大脑的层和区域。

视频: Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain&#39;s macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury.

健康和受伤斑马鱼脑的大型扫描透射电子显微镜（Nanotomy）

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

科研

发育生物学

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

神经科学

Assessment of Ultrastructural Neuroplasticity Parameters After In Utero Transduction of the Developing Mouse Brain and Spinal Cord

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ultrastructural parameters in unambiguously identified topographical structures, affected by a protein of interest that is introduced into the organism via viral transfer. This combined approach allows for a smooth transition from macrostructural to ultrastructural identification by following topographical navigation maps in a tissue atlas. High-resolution electron microscopy of the in-utero-transduced tissue reveals the fine ultrastructure of the neuropil and its plasticity parameters, such as cross-sectioned synaptic bouton areas, the number of synaptic vesicles and mitochondria within a bouton profile, the length of synaptic contacts, cross-sectioned axonal areas, the thickness of myelin sheaths, the number of myelin lamellae, and cross-sectioned areas of mitochondria profiles. The analysis of these parameters reveals essential insights into changes of ultrastructural plasticity in the areas of the nervous system that are affected by the viral transfer of the genetic construct. This combined method can not only be used for studying the direct effect of genetically engineered biomolecules and/or drugs on neuronal plasticity but also opens the possibility to study the in utero rescue of neuronal plasticity (e.g., in the context of neurodegenerative diseases).

The combination of transmission electron microscopy and in utero transduction is a powerful approach for studying morphological changes in the fine ultrastructure of the nervous system during development. This combined method allows deep insights into changes in structural details underlying neuroplasticity with respect to their topographical representation.

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

The present study combines in utero transduction with transmission electron microscopy (TEM) aiming at a precise morphometrical analysis of ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发​​生与再生

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Immunostaining of Biocytin-filled and Processed Sections for Neurochemical Markers

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

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

对于神经化学标记用biocytin填充和加工部分的免疫染色

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

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 ...

Biocytin Recovery and 3D Reconstructions of Filled Hippocampal CA2 Interneurons

How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described here identifies the basic building blocks of this circuitry. The in-depth studies of cortical regions will ultimately provide other scientists with the circuit components needed for an understanding of how the brain acquires, processes and stores information and what goes wrong in disease, while the electrophysiological and morphological data are widely used by computational neuroscientists in the construction of model networks that explore information processing. The protocol outlined here describes how biocytin-filled cells recorded in the CA2 region of the hippocampus are recovered and then reconstructed in&#160;3D. Additionally, the protocol describes the demonstration of calcium binding protein or peptide content in recorded interneurons.

The protocol outlined here describes the immunofluorescence analysis, biocytin recovery and high-quality reconstructions of hippocampal CA2 interneurons following the intracellular electrophysiological recordings in vitro, allowing neuronal characterization and ultimately fine neuronal anatomy to be studied.

填充海马 ca2 间神经元的生物环素恢复与三维重建

How cortical network activity processes information is of importance to a large number of basic and clinical scientific questions. The protocol described ...

Localization of the Locus Coeruleus in the Mouse Brain

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional abnormalities of LC impact several brain regions including cortex, hippocampus, and cerebellum and may contribute to depression, bipolar disorder, anxiety, as well as Parkinson disease and Alzheimer disease. These disorders are often associated with metal misbalance, but the role of metals in LC is only partially understood. Morphologic and functional studies of LC are needed to better understand the human pathologies and contribution of metals. Mice are a widely used experimental model, but the mouse LC is small (~0.3 mm diameter) and hard to identify for a non-expert. Here, we describe a step-by-step immunohistochemistry-based protocol to localize the LC in the mouse brain. Dopamine-&#946;-hydroxylase (DBH), and alternatively, tyrosine hydroxylase (TH), both enzymes highly expressed in the LC, are used as immunohistochemical markers in brain slices. Sections adjacent to LC-containing sections can be used for further analysis, including histology for morphological studies, metabolic testing, as well as metal imaging by X-ray fluorescence microscopy (XFM).

The locus coeruleus is a small cluster of neurons involved in a variety of physiological processes. Here, we describe a protocol to prepare mouse brain sections for studies of proteins and metals in this nucleus.

小鼠大脑中耳蜗的定位

The locus coeruleus (LC) is a major hub of norepinephrine producing neurons that modulate a number of physiological functions. Structural or functional ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

利用突触锌组织化学在发育和成人大脑中揭示不同的区域和叶片

摘要

探索更多视频

此视频中的章节

健康和受伤斑马鱼脑的大型扫描透射电子显微镜（Nanotomy）

钙成像的急性下区片的制备

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发生与再生

在脑切片检测轴突本地化mRNAs的高空间分辨率

对于神经化学标记用biocytin填充和加工部分的免疫染色

海马鼠脑切片的突触密度评价

填充海马 ca2 间神经元的生物环素恢复与三维重建

小鼠大脑中耳蜗的定位

新生大鼠脑组织在神经端子突形圆锥花序分布超微结构形态分析中的制备

利用突触锌组织化学在发育和成人大脑中揭示不同的区域和叶片

摘要

探索更多视频

此视频中的章节

健康和受伤斑马鱼脑的大型扫描透射电子显微镜（Nanotomy）

钙成像的急性下区片的制备

小鼠脑脊髓子宫转导后超微结构神经可塑性参数的评价

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发​​生与再生

在脑切片检测轴突本地化mRNAs的高空间分辨率

对于神经化学标记用biocytin填充和加工部分的免疫染色

海马鼠脑切片的突触密度评价

填充海马 ca2 间神经元的生物环素恢复与三维重建

小鼠大脑中耳蜗的定位

新生大鼠脑组织在神经端子突形圆锥花序分布超微结构形态分析中的制备

斑马鱼成人端脑的一刀，受伤:一个方法调查脊椎动物脑部神经发生与再生