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>

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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

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

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

监测变化的细胞内钙离子浓度和突触效能的软体动物<em&gt;海兔</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

科研

神经科学

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

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

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

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

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

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

从成年鼠脑的常规和非常规地区日益增长的神经干细胞

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to na&#239;ve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. 
High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/na&#239;ve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

The identification of molecules and pathways controlling synaptic plasticity and memory is still a major challenge in neuroscience. Here, a workflow is described addressing the relative quantification of synaptic proteins supposedly involved in the molecular reorganization of synapses during learning and memory consolidation in an auditory learning paradigm.

鼠脑区的高分辨率定量突触蛋白质双向听觉辨别学习后

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Exploring Deep Space - Uncovering the Anatomy of Periventricular Structures to Reveal the Lateral Ventricles of the Human Brain

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this challenging. Because the ventricles are negative spaces located deep within the brain, the only way to understand their anatomy is by appreciating their boundaries formed by related structures. Looking at a 2D representation of these spaces, in any of the cardinal planes, will not enable visualisation of all of the structures that form the boundaries of the ventricles. Thus, using 2D sections alone requires students to compute their own mental image of the 3D ventricular spaces. The aim of this study was to develop a reproducible method for dissecting the human brain to create an educational resource to enhance student understanding of the intricate relationships between the ventricles and periventricular structures. To achieve this, we created a video resource that features a step-by-step guide using a fiber dissection method to reveal the lateral and third ventricles together with the closely related limbic system and basal ganglia structures. One of the advantages of this method is that it enables delineation of the white matter tracts that are difficult to distinguish using other dissection techniques. This video is accompanied by a written protocol that provides a systematic description of the process to aid in the reproduction of the brain dissection. This package offers a valuable anatomy teaching resource for educators and students alike. By following these instructions educators can create teaching resources and students can be guided to produce their own brain dissection as a hands-on practical activity. We recommend that this video guide be incorporated into neuroanatomy teaching to enhance student understanding of the morphology and clinical relevance of the ventricles.

This paper demonstrates the effective use of a fiber dissection method to reveal the superficial white matter tracts and periventricular structures of the human brain, in three-dimensional space, to aid student comprehension of ventricular morphology.

探索深空-揭开人脑侧脑室的脑室结构解剖

Anatomy students are typically provided with two-dimensional (2D) sections and images when studying cerebral ventricular anatomy and students find this ...

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

光遗传学与人工 microRNAs 相结合对完整神经元回路中基因击倒对突触前功能的影响

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

免疫荧光定量小鼠脑突触蛋白的异种分布

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

Non-Invasive Modulation and Robotic Mapping of Motor Cortex in the Developing Brain

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries unique challenges in children. Similarly, transcranial direct current stimulation (tDCS) can improve motor learning in adults but has only recently been applied to children. The use of tDCS and emerging techniques like high&#8211;definition tDCS (HD-tDCS) require special methodological considerations in the developing brain. Robotic TMS motor mapping may confer unique advantages for mapping, particularly in the developing brain. Here, we aim to provide a practical, standardized approach for two integrated methods capable of simultaneously exploring motor cortex modulation and motor maps in children. First, we describe a protocol for robotic TMS motor mapping. Individualized, MRI-navigated 12x12 grids centered on the motor cortex guide a robot to administer single-pulse TMS. Mean motor evoked potential (MEP) amplitudes per grid point are used to generate 3D motor maps of individual hand muscles with outcomes including map area, volume, and center of gravity. Tools to measure safety and tolerability of both methods are also included. Second, we describe the application of both tDCS and HD-tDCS to modulate the motor cortex and motor learning. An experimental training paradigm and sample results are described. These methods will advance the application of non-invasive brain stimulation in children.

We demonstrate protocols for the modulation (tDCS, HD-tDCS) and mapping (robotic TMS) of the motor cortex in children.

发育中大脑运动皮质的非侵入性调制和机器人映射

Mapping the motor cortex with transcranial magnetic stimulation (TMS) has potential to interrogate motor cortex physiology and plasticity but carries ...