作者感谢 Keali hm、路易莎 Frieß和海克 Menzel 的专家技术援助;海尔 Brünner 的动物护理;和 Chistopher s 病房为 ImageJ 软件的光 disector 网格插件的世代和发行。

对动物的照顾和使用的所有适用的国际、国家和/或机构准则都得到遵守。该议定书由德国 Wuerzburg Regierung Unterfranken 的地方当局批准. 
 1. 组织处理和免疫组化 <ol> <li> 安乐与 CO 2 或任何其他已批准方法的小鼠. </li>
	<li> 灌注六12周老 C56Bl/6N 雄性小鼠 transcardially 10 毫升0.1 米的磷酸盐缓冲盐水 (pbs) 使用25克针和输液泵, 其次70毫升4% 甲醛 (粉煤灰) 在 0.1 M PBS. 
	 警告: 煤灰是有毒、过敏和致癌的. </li>
	<li> 在 +0.74 mm Bregma (图 25, Paxinos 和富兰克林鼠脑图谱 14 ) 的区域内, 用大脑矩阵切片器解剖在日冕平面上的小鼠大脑. </li>
	<li> 将大脑的背侧部分, 包括 SN, 在0.1 米 PBS 中的4% 粉煤灰中, 1 d 在4和 #176; 浸泡固定的 C. </li>
	<li> 将30% 蔗糖/0.1 摩尔 PBS 溶液交换为低温保护, 并在4和 #176 中孵育另 2 d; C. </li>
	<li> 将大脑的背部部分放入一个 cryomold 填充最佳切削温度 (OCT) 化合物, 并在液态干冰冷却戊中缓慢冻结组织. 
	 警告: 干冰非常冷 (-78 和 #176; C);使用个人防护设备 (PPE), 包括手套, 同时处理干冰。干冰在 CO 2 中蒸发;因此, 在通风罩下工作. </li>
	<li> 连续剪切 30 #181; m 在冠状面上的冰冻切片, 并收集剖面, 从 Bregma (图 51, Paxinos 和富兰克林鼠脑图谱 14 ) 起2.46 毫米开始, 在4.04 毫米从 Bregma (图 64, Paxinos 和富兰克林鼠脑图谱 14 ). </li>
	<li> 存储四系列的管中充满剂 (30% 甘油, 30% 乙二醇, 40% PBS) 在-20 和 #176;在切片过程中, 通过在脑干的上部区域穿刺一个小孔来标记右半球. </li>
	<li> 对于免疫组织化学染色, 每只老鼠选择一组切片。Preincubate 自由漂浮部分1小时在10% 正常山羊血清 (NGS)/2% BSA/0.5% 洗涤剂在0.1 摩尔 PBS 在一个振动筛上。用兔 anti-mouse 酪氨酸羟化酶 (TH; 1:1, 000 稀释) 在0.1 摩尔 PBS 中稀释 2% NGS/2%bsa/0.5% 洗涤剂, 在4和 #176; C. </li>
	<li> 在室温下应用二次抗体对抗家兔 (1:100 稀释) 2 小时, 其次是素/生物素试剂 (1:100 稀释)。孵育和染色 33-diaminobenzidine-tetrahydrochloride (民建联) 和 H 2 O 2 。在涂层对象幻灯片上着色后, 将其装入这些部分. 
	注: TH + 多巴胺能 SN 神经元在 图 1a 中显示。通过染色一个系列, 平均 13 sn 部分, 从侧延伸到尾部部分的 sn 致密 (SNpc) 和福橘, 染色每动物 ( 图 1b )。各节以120和 #181 分隔; m (1/4 系列) ( 图 1c ). 
	 警告: 民建联是一种可疑的致癌物。它是有毒的接触和吸入。在与民建联合作时使用 PPE. </li>
</ol> 2。获取图像 <ol> <li> 使用与显微镜耦合的图像软件捕获 TH 免疫着色的 SN 剖面。分别分析一个系列的每个部分. </li>
	<li> 使用图像处理软件的扫描幻灯片选项。以 TIFF 格式保存高质量图像 ( 图 2 ). 
	注意: 图像分辨率是每 cm 312 像素. </li>
	<li> 按 #34; 获取和 #34; 打开获取窗口 ( 图 2a ). </li>
	<li> 将分设置为和 #34; 2 和 #34; 用于灰度图像. 
	注意: 获取灰度图像而不是彩色图片会减小最终堆栈图像文件的大小 ( 图 2a ). </li>
	<li> 单击并 #34; 显示实况和 #34; 在获取窗口中打开一个新窗口, 其中显示该节的实时图像 ( 图 2 a 和 b ). </li>
	<li> 单击并 #34; 舞台和 #34; 在获取窗口中。选择和 #34; 扫描幻灯片和 #34; 在下拉菜单中检查该框 ( 图 2c ). 
	注意: 这使得在 x、y 平面上的高度放大的 SN 图像 (630X 放大) 的获取和对准, 以及在1和 #181 的连续图像之间的距离的堆栈图像的获取; m 在 z 平面中, 覆盖总范围为13和 #181;m. </li> <li> 选择2.5X 目标以搜索 SN. </li>
	<li> 将目标更改为 63x. </li>
	通过单击和 #34, <li> 定义该节的左上角 (TopLeft) ( 图 2c ) 和右下角 (LowRight) ( 图 2d ); 设置. 和 #34; </li>
	<li> 单击并 #34; z 系列和 #34; 检查 #34; z 平面系列和 #34; 框 ( 图 2e ). </li>
	<li> 使用成像软件确定每节三随机选取的计数点的实际安装厚度。为此, 请单击 #34; 查看顶部偏移量和 #34; 若要定义节的顶部 ( 图 2e ), 然后单击并 #34; 停止查看顶部和 #34; ( 图 2f )。然后, 点击和 #34; 查看底部偏移量和 #34; ( 图 2f ), 然后单击并 #34; 停止查看底部和 #34; ( 图 2g )。记下剖面的粗细. </li>
	<li> 从顶部偏移量中减去3和 #181; m (保护区域), 并将结果键入顶部偏移槽 ( 图 2h )。从顶部偏移数减去13和 #181; m (光学 disector 的高度), 并将结果键入底部偏移槽 ( 图 2i )。选择以下参数: 步骤, 14;大小, 1.00 ( 图 2i ). 
	注: 这对应于13和 #181 的 z 平面上的厚度; m, 具有1和 #181; 连续图像之间的距离. </li>
	<li> 选择要保存文件的目录 ( 图 2j ). </li>
	<li> 单击并 #34; 序列和 #34; 启动获取 ( 图 2j ). </li>
	<li> 单击 #34;P 加工和 #34, 然后选择以下参数: #34; 所有飞机、#34、#34、序列、#34、#34、蒙太奇、#34; 和 #34; 快速和 #34; 和 #34; 拼接和 #34; 框, 图像重叠为 10% ( 图 2k )。开始通过点击和 #34 来缝合图像; 开始和 #34;. 
	注意: 这将生成用于分析的堆栈图像 ( 图 2 l, 图 3a e )。
</ol> 3。视学评估序列 <ol> <li> 使用 NIH ImageJ 软件版本4.7 执行对 SN 堆栈图像的分析. </li>
	<li> 下载 ImageJ.nih.gov 网站所需的两个插件: #34; 网格叠加和 #34; 插件 15 和 #34; 单元计数器和 #34; 插件 16 。按所述安装两个插件. </li>
	<li> 对于无偏视学评估, 请按如下所示定义计数参数: 网格大小、130 x 130 和 #181; 计数帧、50 x 50 和 #181; m; 和光学 disector 高度, 13 和 #181; m. </li>
	<li> 通过单击 #34 打开图像; 文件和 #34; #160; #8594; #34; 打开和 #34; ( 图 4a ) 通过使用和 #34; 分析与 #34; #160; #8594; #34; 设置缩放和 #34; 命令 ( 图 4b )。对于此步骤, 请将定义的大小从像素更改为和 #181; m ( 图 4c ). 
	注意: 对于分析的图像, 425 像素对应于100和 #181; m. </li>
	<li> 选择 #34;P lugins 和 #34; #160; #8594; #34; 网格和 #34; #160; #8594; #34; 网格类型: 线和 #34; 命令随机插入网格。检查 #34; 随机偏移和 #34; 框。定义网格中一个正方形的大小 (网格正方形) 为130和 #181; m x 130 和 #181; m = 16900 和 #181; m 2 ( 图 3b 和 图 4d 和 e ). </li>
	<li> 将图像类型从16位更改为 RGB 颜色 (单击和 #34; 图像和 #34; #160; #8594; #34; 类型和 #34; #160; #8594; #34; rgb 颜色和 #34;) ( 图 4f )。找到 SNpc, 如 Baquet et al. 所述 17 . 将 SNpc 与 #34;P aintbrush 工具和 #34 包围; (画笔宽度:11) ( 图 3c 和 图 4g i ). </li>
	<li> 撤消和 #34; 设置缩放和 #34; 命令通过单击和 #34; 分析和 #34; #8594; #34; 设置规模和 #34; #8594; #34; 单击以删除缩放和 #34; ( 图 4j -l ) 以像素为单位启用计算的性能, 而比和 #181; m. </li>
	<li> 制作截图 ( 图 4m ), 保存图像文件, 然后用 ImageJ ( 图 4n ) 打开新文件。选择和 #34;P 点和 #34; ( 图 4o ) 和画笔宽度 25 ( 图 4p )。标记与 SN 联系的每个网格正方形, 以放置一个光学 disector ( 图 4q )。对在所述 SN 中完全或部分局部化的所有光学 disectors 中的单元格数进行分析. </li>
	<li> 选择一个包含 SN 部分的网格正方形。用 ImageJ 中的光标测量此正方形的左上角, 以定义要开始的 x 和 y 坐标 ( 图 4r ). 
	注: 使用 #34; 光学 disector 位置和 #34; ( 图 4s ), 如步骤4所述, 将为光学 disector 定位插入坐标. </li>
	<li> 计算光学 disector 位置的方法是使用 "电子表格模板", #34; 光学 disector 位置, #34; 如步骤4所述 </li>
	<li> 校准光学 disector (由克里斯托弗· s 沃德编写的宏, 补充文件 ) 通过单击 #34;P lugins 和 #34; #8594; #34; 宏和 #34; #8594; #34; 编辑和 #34; 图 4t ), 打开和 #34; opt_dis_grid.txt 和 #34; 文件 ( 图 4u ), 并定义 #34 的大小; usergrid 和 #34; 以像素为单位, 与光学 disector ( 图 4v ) 的 x、y 维度相对应。选择大小为50和 #181; m x 50 和 #181; m, 使 usergrid 的一侧的大小定义为212.5 像素 (50 x 4.25 像素). </li>
	<li> 通过插入定义光学 disector ( 图 4v ) 中心的 ImageX 和 ImageY 坐标来确定光盘 disector 在堆栈图像中的位置。运行宏 (#34; 宏和 #34; 和 #8594; #34; 运行宏和 #34;) ( 图 4w ). 
	注意: 网格将从堆栈图像 ( 图 3d 和 图 4x ) 中消失。在 z 平面 ( 图 3e 和 图 4x ) 中向下移动时, 光学 disector 将持续. </li>
	<li> 选择和 #34;P lugins 和 #34; #160; #8594; #34; 单元格计数器和 #34 启动单元计数器插件 ( 图 4y )。初始化单元格计数器并选择一个标记类型 ( 图 4z )。放大图像 (点击和 #34; + 和 #34;) 和执行视学评估的细胞数放大 200%. </li>
	<li> 通过圆形或卵球形的形状和细胞大小来识别多巴胺能神经元 perikarya ( 图 1a )。使用视学规则量化单元格的数量. 
	注意: 由于光学 disector 是一个3维多维数据集 ( 图 5a ), 因此将排除边定义为多维数据集的前、左和上侧 (红色)。因此, don 和 #39, 不要计算与红线 (左和前) 或顶部接触的 TH 细胞。将单元格计数的结果添加到已准备好的电子表格文件中, #34; 计算单元计数和 #34; (步骤 5). </li>
	<li> 根据 SN 图像, 计算下一个网格正方形, 用于将光学 disector 用于下一单元格计数, 使用 x、y 步骤覆盖上覆网格的大小 ( 图 5b ) 和 #34; 光学 disector 位置和 #34;电子表格模板, 如步骤4所述. </li>
	<li> 使用 and #34 对单元格数进行估计; 计算单元计数和 #34; 电子表格 (步骤 5). </li>
</ol> 4。光学 Disector 位置的计算 #34; 光学 Disector 位置和 #34; 电子表格模板 <ol> <li> 计算每个光学 Disector 的大小和位置, 将其放置在 SN 轮廓内的上覆盖网格中 ( 图 5b )使用和 #34; 光学 disector 位置. xlsx 和 #34; 电子表格文件 ( 补充文件 ). </li>
	<li> 插入按 #34 定义的每 #181 像素的转换; 设置缩放和 #34; 命令, 进入和 #34 的黄色标记位置; 光学 disector 位置. xlsx 和 #34; 文件 ( 图 6a 和 补充文件).</li>
	<li> 插入 disector 的计划大小和上覆网格的网格正方形的大小, 由一侧 (在和 #181; m) 的长度定义, 到橙色标记的位置. 
	注意: 结果在橙色方框旁边的红色方框中描述. </li>
	<li> 从作为起点的 SN 轮廓中的网格正方形开始 (在上面的步骤3.9 中选择一个网格正方形作为开始). </li>
	<li> 将 x 和 y 坐标 (如步骤3.9 所示) 插入文件的绿色框中 (通过使用这些值, 计算第一个正方形内的光学 disector 中心的 x、y 坐标, 并在蓝色框中描述结果)。运行 opt_dis_grid 的. txt 宏 ( 补充文件 ). </li>
	<li> 相对于第一个网格正方形 (棕色框), 通过插入网格平方距离 (以平方数度量) 来计算连续光学 disectors 的 x、y 坐标. 
	注: + x: 方格位于右侧;-x: 网格正方形位于左边;+ y: 网格正方形位于底部;-y: 网格广场位于顶端。结果显示在灰色框中. </li>
</ol> 5。使用 and #34 估计单元格数; 计算单元数和 #34; 电子表格 <ol> <li> 使用已发布的公式计算每个动物 (N) 的估计数 + SN 单元格个数由西部 et al ., 1991 3 , 在哪里和 #8721; Q - 定义为在一个脑部的所有光学 disectors 中计数的 TH + 神经元总数. 
	注意: 该方程中包含了光学 disector (h) 与截面 (t) 的测量厚度有关的高度。区域取样分数 (asf) 定义为在网格的平方大小 (a x, 光学 disector) 帧大小的光 disector (一个光学的) 的面积 (a) 的比例 (y 步) (光学 disector/x、y 步) ( 图 3b ) 和切片取样分数是指整个序列切脑部分的比例: <img alt="Equation 1" src="/files/ftp_upload/56103/56103eq1.jpg"/> 以确保高质量的定量估计, 系数错误 (CE) 应低于0.10。通过 Keuker et al 来确定 CE 的使用公式, 2001 10 : <img alt="Equation 2" src="/files/ftp_upload/56103/56103eq2.jpg"/> </li> <li> 视学估计每个鼠标, 插入有关每个鼠标生成的系列总数的指示信息, 光学 disector 的高度, 光学 disector 的 x、y 区域 (光学 disector, 在和 #181; m 2 ), 网格正方形的 x、y 区域 (x、y 步, #181; m 2 ), 以及使用 and #34 的黄色框中的截面 (#181; m) 的平均测量厚度; 计算单元数. xlsx 和 #34; 电子表格文件 ( 图 6b 和补充文件 ).</li>
	<li> 从同一动物中分析每个 SN 段, 如步骤5所述. </li>
	<li> 在灰色框中插入每个光学 disector 的单元格计数, 每一行引用一个 SN 节, 使用 and #34; 计算单元计数. xlsx 和 #34; 电子表格文件 ( 补充文件 ). 
	注意: 在蓝色方框 ( 图 6b ) 中描述了估计的单元数和计算出的 CE. </li>
</ol> 6。使用商业上可用的视学系统估计单元格数 <ol> <li> 通过单击 #34;P 长袍和 #34 开始估计多巴胺能 SNpc 细胞数; #8594; #34; 光学分馏塔工作流和 #34; 打开光学分馏塔工作流窗口. 
	注意: 将弹出一个新窗口, 以确定是否要计算新的 SN 系列. </li>
	<li> 通过单击和 #34 启动新的 "TH + SN" 系列; 开始新的主题和 #34; 在弹出窗口中. </li>
	<li> 遍历光分馏塔工作流的不同步骤。在第一步中设置主题。为此, 请插入调查员和 #39 的名称、主题和 #39; s 名称 (SN 组) 以及其他有用的信息。插入此 SN 要计数的节数。插入切口组织切片的厚度。插入节间隔, 在本例中为四。插入随机确定的节编号以开始. </li>
	<li> 在第二步中, 将显微镜设置为低放大率 (2X 目标), 并选择和 #34; 2X Mag 透镜和 #34; 从菜单中. </li>
	<li> 在步骤3中, 用鼠标左键跟踪感兴趣的区域, 这是 SNpc, 如 Baquet et al ., 2009 17 . </li>
	<li> 在步骤4中将显微镜设置为高倍放大倍数。为此, 将目标更改为 100X, 然后选择和 #34; 100X Mag 透镜和 #34; 从下拉菜单中. </li>
	<li> 通过聚焦到节的顶部和底部来测量步骤5中的已装入厚度. </li>
	<li> 在步骤6中, 将计数帧大小定义为 50 x 50 和 #181; m; 这是光学 disector (x、y) 的大小. </li>
	<li> 在步骤7中, 将系统随机抽样 (SRS) 网格的大小设置为 130 x 130 和 #181; m. </li>
	<li> 在步骤8中输入3和 #181; m 保护区域. </li>
	<li> 在步骤9中保存设置 </li>
	<li> 最后, 在光学分馏塔工作流的步骤10中, 计算 SRS 网格中每个光学 disectors 中的 TH + 多巴胺能细胞。为此, 请先点击 #34; 计数和 #34; 按钮. </li>
	<li> 完成一节后, 单击 #34; 开始下一节和 #34; 按钮以启动同一 sn 系列的下一个 sn 段的光学分馏塔工作流. </li>
	<li> 当一个 sn 系列的最后一个 sn 部分被量化后, 单击 #34; 我和 #39; 我已经完成计数. #34; </li>
	<li> 单击 #34; 查看结果和 #34; 按钮以显示视学采样的结果. </li>
</ol>

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+proliferation+following+stroke+in+the+mouse+depends+on+distance+from+the+infarct.">Astrocyte proliferation following stroke in the mouse depends on distance from the infarct.</a> PLoS One. 6 (11), e27881 (2011).</li><li>Robinson, S., 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=Microstructural+and+microglial+changes+after+repetitive+mild+traumatic+brain+injury+in+mice.">Microstructural and microglial changes after repetitive mild traumatic brain injury in mice.</a> J Neurosci Res. 95 (4), 1025-1035 (2017).</li><li>Paxinos, G., Franklin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. , (2001).</li><li>. Grid_Overlay.java Available from: <a target="_blank" href="https://imagej.nih.gov/ij/plugins/graphic-overlay.html">https://imagej.nih.gov/ij/plugins/graphic-overlay.html</a> (2010)</li><li>. cell_counter.jar Available from: <a target="_blank" href="https://imagej.nih.gov/ij/plugins/cell-counter.html">https://imagej.nih.gov/ij/plugins/cell-counter.html</a> (2010)</li><li>Baquet, Z. C., Williams, D., Brody, J., Smeyne, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+model-based+(2D)+and+design-based+(3D)+stereological+methods+for+estimating+cell+number+in+the+substantia+nigra+pars+compacta+(SNpc)+of+the+C57BL/6J+mouse.">A comparison of model-based (2D) and design-based (3D) stereological methods for estimating cell number in the substantia nigra pars compacta (SNpc) of the C57BL/6J mouse.</a> Neuroscience. 161 (4), 1082-1090 (2009).</li><li>Fu, Y., 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=A+cytoarchitectonic+and+chemoarchitectonic+analysis+of+the+dopamine+cell+groups+in+the+substantia+nigra,+ventral+tegmental+area,+and+retrorubral+field+in+the+mouse.">A cytoarchitectonic and chemoarchitectonic analysis of the dopamine cell groups in the substantia nigra, ventral tegmental area, and retrorubral field in the mouse.</a> Brain Struct Funct. 217 (2), 591-612 (2012).</li><li>German, D. C., Manaye, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Midbrain+dopaminergic+neurons+(nuclei+A8,+A9,+and+A10):+three-dimensional+reconstruction+in+the+rat.">Midbrain dopaminergic neurons (nuclei A8, A9, and A10): three-dimensional reconstruction in the rat.</a> J Comp Neurol. 331 (3), 297-309 (1993).</li><li>Smeyne, R. J., 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=Assessment+of+the+Effects+of+MPTP+and+Paraquat+on+Dopaminergic+Neurons+and+Microglia+in+the+Substantia+Nigra+Pars+Compacta+of+C57BL/6+Mice.">Assessment of the Effects of MPTP and Paraquat on Dopaminergic Neurons and Microglia in the Substantia Nigra Pars Compacta of C57BL/6 Mice.</a> PLoS One. 11 (10), e0164094 (2016).</li></ol>

c.w.i. 为 Merz 制药、LLC 和 TEVA 的科学委员会服务;从易普森、Merz 制药、LLC 和 Allergan、Inc. 的旅费中获得资金;并已收到 Merz、TEVA、Allergan、Inc. 外的演讲者酬金。企业曾担任波士顿科学、敦和 AbbVie 的顾问, 并获得敦、波士顿科学、AbbVie、比亚尔、Allergan 和 GlobalKinetics 在提交的工作之外的酬金。

视学从组织加工开始。在视学分析过程中, 必须小心地进行 SN 组织的连续切割, 以防止节段丢失。另外, 一个重要的步骤是标记一个半球, 以便在执行视学时区分左 SN 的右侧。在脑干上部放置一个小孔, 在所提出的研究中产生了最好的结果。此外, 由于使用光学分馏塔方法, 要求组织被切割在厚的部分约30-40 µm, 而不是5-10 µm 更常用的免疫组化, 抗体和其他培养液组织渗透期间免疫组织化学染色必须?...

神经元细胞数的定量是临床前帕金森病研究的关键, 以确定黑质 (SN)1,2内神经的水平。对感兴趣区域中的单元格数的无偏视学估计被认为是金标准3、4、5。
在无偏视学的出现之前, 部分神经元的数量通过操纵计数的细胞剖面来进行评估, 以纠正神经元在一节中进入视线的可变概率。最常用的方法之一是修正由6所描述的量化单元计数。如果在相邻的薄片中发现同一单元格的碎片, 则此方法试图考虑到单元格可以多次量化。因此, 在计算单元的形状、大小和方向方面需要假设的公式7,8。但是, 这些假设通常没有实现, 因此导致了系统的错误和与实际单元格数 (即偏倚) 的背离。此外, 还不能通过附加采样3来减少偏差。
对于使用光学分馏塔的细胞数的视学估计, 应用数学原理直接在定义的3维体积中直接估计细胞数。这种方法的优点是它不涉及被计算单元的形状、大小和方向的假设。因此, 估计的单元格数更接近于真实值, 并且随着样本大小的增加 (即,无偏)3变得更接近。由于使用视学保持方法无偏时必须遵循许多规则, 因此已开发现成的商业视学系统 (供审查, 请参阅 20054)。专门的视学系统实现了设计视学方法与先验定义的探针和抽样方案的视学评估, 导致独立的形状, 大小, 空间分布, 和方向要分析的单元格4,9。然而, 商业上可用的视学系统是昂贵的;这可能会限制新研究的实施。
本研究的目的是开发一种可用的技术设计视学估计多巴胺能细胞计数在小鼠 SN, 采用光学分馏塔方法和使用标准显微镜设备 (即,光显微镜, 标准显微镜软件, 和机动的 x, y, z 阶段)。为此, 提出了如何处理小鼠脑组织和如何使用设计无偏视学估计 SN 细胞数的 step-by 步骤指南。此外, 还提供了计算估计单元数和误差系数 (CE) 的模板。
这里描述的方法不限于对 SN 的分析, 但可以适应在其他的解剖定义的老鼠或老鼠大脑区域使用。例如, 无偏视学被用来估计海马体的神经元细胞数10和轨迹斑11。此外, 还可以评估除神经元以外的其他细胞类型, 如星状细胞12和小胶质红细胞13。因此, 这种方法对于打算在研究中实现无偏视学的科学家是有用的, 但他们不愿意花很多钱购买视学系统。

<table><tbody><tr><td>Paxinos mouse atlas</td><td></td><td></td><td>The Mouse Brain George Paxinos Keith B.J.FranklinCopyright @2001 by Academic Press CD Rom designet &amp;amp; created by Paul Halasz</td></tr><tr><td>brain matrix slicer mouse</td><td>Zivic Instruments</td><td>BSMAS 001-1</td><td></td></tr><tr><td>paraformaldehyde</td><td>Merck</td><td>1040051000</td><td></td></tr><tr><td>sucrose /D(+) Saccharose</td><td>Roth</td><td>4621.1</td><td></td></tr><tr><td>isopentane</td><td>Roth</td><td>3927.1</td><td></td></tr><tr><td>glycerol</td><td>Merck</td><td>1040931000</td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>32205-1L</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;phosphate buffered saline ingredients:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>sodium chloride</td><td>Sigma Aldrich</td><td>31434-1KG-R</td><td></td></tr><tr><td>potassium dihydrogen phosphate</td><td>Merck</td><td>1048731000</td><td></td></tr><tr><td>di-sodium hydrogen phosphate dihydrate</td><td>Merck</td><td>1065801000</td><td></td></tr><tr><td>potassium chloride</td><td>Merck</td><td>1049360500</td><td></td></tr><tr><td>normal goat serum</td><td>Dako</td><td>X0907</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A4503-100G</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>X100-100ml</td><td>detergent</td></tr><tr><td>3,3-Diaminobenzidine-tetrahydrochlorid/DAB tablets 10mg pH 7.0</td><td>Kem En Tec</td><td>4170</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;/ Hydrogen peroxide 30%</td><td>Merck</td><td>1072090250</td><td></td></tr><tr><td>avidin/biotin reagent</td><td>Thermo Scientific</td><td>32050</td><td>Standard Ultra Sensitive ABC Staining Kit, 1:100</td></tr><tr><td>rabbit anti mouse tyrosine hydroxylase antibody</td><td>abcam</td><td>Ab112</td><td>1:1000</td></tr><tr><td>biotinylated goat-anti-rabbit IgG H+L</td><td>vector laboratories</td><td>BA-1000</td><td>1:100</td></tr><tr><td>StereoInvestigator version 11.07</td><td>MBF</td><td></td><td></td></tr><tr><td>BX53 microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>Visiview</td><td>Visitron Systems GmbH</td><td>3.3.0.2</td><td></td></tr><tr><td>Axiophot2</td><td>Zeiss</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>NIH</td><td>Version 4.7</td><td></td></tr><tr><td>Tissue-TEK OCT</td><td>Sakura</td><td>4583</td><td></td></tr><tr><td>dry ice</td><td></td><td></td><td></td></tr><tr><td>grid overlay plugin</td><td>Wayne Rasband</td><td></td><td>https://imagej.nih.gov/ij/plugins/graphic-overlay.html</td></tr><tr><td>cell counter plugin</td><td>Kurt de Vos</td><td></td><td>https://imagej.nih.gov/ij/plugins/cell-counter.html).&amp;nbsp;</td></tr><tr><td>optical disector macro</td><td>Christopher Ward</td><td></td><td></td></tr><tr><td>C57Bl/6N male mice</td><td>Charles River, Germany</td><td></td><td></td></tr><tr><td>SuperFrost Plus coated object slides</td><td>Langenbrinck, Germany</td><td></td><td></td></tr><tr><td>25G needle Microlance 3</td><td>BD</td><td>300400</td><td></td></tr><tr><td>REGLO Analog Infusion pump</td><td>Ismatec</td><td>ISM 829</td><td></td></tr><tr><td>StereoInvestigator system</td><td></td><td></td><td>StereoInvestigator version 11.07</td></tr><tr><td>BX53 microscope</td><td></td><td></td><td>BX53 microscope</td></tr><tr><td>self-assorted stereology</td><td></td><td></td><td>Visiview</td></tr><tr><td>Axiophot2</td><td></td><td></td><td>Axiophot2</td></tr></tbody></table>

stereological estimation of dopaminergic neuron number in the mouse substantia nigra using the optical fractionator and standard microscopy equipment

在临床前帕金森病的研究中, 对体道的分析, 包括对黑质中多巴胺能神经元损失的定量, 是必不可少的。为了估计总的多巴胺能神经元数, 采用光分馏法的无偏视学目前被认为是金标准。因为光分馏塔方法的理论是复杂的, 因为视学是难以实现的, 没有专门的设备, 一些商业上可用的完整的视学系统, 包括必要的软件确实存在, 纯粹细胞计数的原因。由于购买专门的视学设置并不总是可行的, 由于许多原因, 本报告描述了使用标准显微镜设备对多巴胺能神经元细胞计数进行视学估计的方法, 包括光学显微镜、带有成像软件的机动对象表 (x、y、z 平面) 和用于分析的计算机。给出了用光学分馏塔法进行视学量化的 step-by 步骤说明, 并给出了计算估计单元数的预编程文件。为了评估这种方法的准确性, 对从商用视学仪器中获得的数据进行了比较。使用此协议和视学设备发现可比较的单元数, 从而证明了该协议对于无偏视学的精确性。

使用所提出的方法, 右 sn 中的 TH + 多巴胺能神经元的估计数介于7363和7987细胞之间, 在左 sn 中, 7446 和7904细胞之间。因此, 平均多巴胺能神经元 (± SEM) 为右 sn 为7647±83细胞, 左 sn 为7675±66。每个动物的计算 CE 值低于 0.08 (范围: 0.073-0. 079) (图 7)。为了确定此方法与商用和专用视学系统的可比性, 还使用视学设置 (图 8) 对 SN 单元数进...

Watch this Scientific Journal Video about Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment at JoVE.

Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

这项工作提出了一个 step-by 步协议的无偏视学估计多巴胺能神经元细胞数在小鼠黑质使用标准显微镜设备 (即,光显微镜, 机动对象表 (x, y, z平面) 和公有域软件进行数字图像分析。

用光学分馏塔和标准显微镜设备对小鼠黑质多巴胺能神经元数的视学估计

In pre-clinical Parkinson&#39;s disease research, analysis of the nigrostriatal tract, including quantification of dopaminergic neuron loss within the ...

stereological-estimation-dopaminergic-neuron-number-mouse-substantia

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

17.9K 次观看.  University Hospital of Würzburg. 这项工作提出了一个 step-by 步协议的无偏视学估计多巴胺能神经元细胞数在小鼠黑质使用标准显微镜设备 (即,光显微镜, 机动对象表 (x, y, z平面) 和公有域软件进行数字图像分析。

视频: Stereological Estimation of Dopaminergic Neuron Number in the Mouse Substantia Nigra Using the Optical Fractionator and Standard Microscopy Equipment

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

科研

神经科学

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, interactions with our environment can induce brain plasticity. Increasingly, research is trying to identify which environments stimulate brain plasticity beneficial for treating brain and behavioral disorders. Two environmental manipulations are described which increase or decrease the number of tyrosine hydroxylase immunopositive (TH+, the rate-limiting enzyme in dopamine (DA) synthesis) neurons in the adult mouse midbrain. The first comprises pairing male and female mice together continuously for 1 week, which increases midbrain TH+ neurons by approximately 12% in males, but decreases midbrain TH+ neurons by approximately 12% in females. The second comprises housing mice continuously for 2 weeks in &#8216;enriched environments&#8217; (EE) containing running wheels, toys, ropes, nesting material, etc., which increases midbrain TH+ neurons by approximately 14% in males. Additionally, a protocol is described for concurrently infusing drugs directly into the midbrain during these environmental manipulations to help identify mechanisms underlying environmentally-induced brain plasticity. For example, EE-induction of more midbrain TH+ neurons is abolished by concurrent blockade of synaptic input onto midbrain neurons. Together, these data indicate that information about the environment is relayed via synaptic input to midbrain neurons to switch on or off expression of &#8216;DA&#8217; genes. Thus, appropriate environmental stimulation, or drug targeting of the underlying mechanisms, might be helpful for treating brain and behavioral disorders associated with imbalances in midbrain DA (e.g. Parkinson&#8217;s disease, attention deficit and hyperactivity disorder, schizophrenia, and drug addiction).

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

中脑多巴胺神经元的数目在成年小鼠环境的调变

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

Reliable Identification of Living Dopaminergic Neurons in Midbrain Cultures Using RNA Sequencing and TH-promoter-driven eGFP Expression

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, leading to bradykinesia, rigidity, and tremor. The identification of living dopaminergic neurons in primary Ventral Mesencephalic (VM) cultures using a fluorescent marker provides an alternative way to study the selective vulnerability of these neurons without relying on the immunostaining of fixed cells. Here, we isolate, dissociate, and culture mouse VM neurons for 3 weeks. We then identify dopaminergic neurons in the cultures using eGFP fluorescence (driven by a Tyrosine Hydroxylase (TH) promoter). Individual neurons are harvested into microcentrifuge tubes using glass micropipettes. Next, we lyse the harvested cells, and conduct cDNA synthesis and transposon-mediated &#34;tagmentation&#34; to produce single cell RNA-Seq libraries1,2,3,4,5. After passing a quality-control check, single-cell libraries are sequenced and subsequent analysis is carried out to measure gene expression. We report transcriptome results for individual dopaminergic and GABAergic neurons isolated from midbrain cultures. We report that 100% of the live TH-eGFP cells that were harvested and sequenced were dopaminergic neurons. These techniques will have widespread applications in neuroscience and molecular biology.

In Parkinson&#39;s Disease (PD), Substantia Nigra (SNc) dopaminergic neurons degenerate, leading to motor dysfunction. Here we report a protocol for culturing ventral midbrain neurons from a mouse expressing eGFP driven by a Tyrosine Hydroxylase (TH) promoter sequence, harvesting individual fluorescent neurons from the cultures, and measuring their transcriptome using RNA-seq.

生活多巴胺能神经元可靠的鉴定中脑培养使用RNA测序和TH-启动子驱动的EGFP表达

In Parkinson&#39;s Disease (PD) there is widespread neuronal loss throughout the brain with pronounced degeneration of dopaminergic neurons in the SNc, ...

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells or structures. These methods have been revolutionary for quantitative analysis of the mammalian brain, in which volumetric cell populations are too high to count manually, and stereology is now the technique of choice whenever estimates of three-dimensional quantities need to be extracted from measurements on two-dimensional sections. All stereological methods are in principle unbiased; however, they rely on proper knowledge about the structure of interest and the characteristics of the tissue. Stereology is based on Systematic Uniformly Random Sampling (SURS), with adjustment of sampling to the most efficient level in respect to precision, providing reliable, quantitative information about the whole structure of interest. Here we present the optical fractionator in conjunction with BrdU immunohistochemistry to estimate the production and survival of newly-formed neurons in the granule cell layer (including the sub-granular zone) of the rat hippocampus following electroconvulsive stimulation, which is among the most potent stimulators of neurogenesis. The optical fractionator technique is designed to provide estimates of the total number of cells from thick sections sampled from the full structure. Thick sections provide the opportunity to observe cells in their full 3-D extent and thus, allow for easy and robust cell classification based on morphological criteria. When correctly implemented, the sensitivity and efficiency of the optical fractionator provides accurate estimates with a fixed and predetermined precision.

Here, we present a stereological method, the optical fractionator, used to quantify the formation of new neurons, and their survival, in the rat hippocampus following electroconvulsive stimulation. When correctly implemented, the sensitivity and efficiency of stereological methods ensures accurate estimates with a fixed and predetermined precision.

用于估计电惊厥治疗大鼠模型中细胞数的光学分级技术

Stereological methods are designed to describe quantitative parameters without making assumptions about size, shape, orientation and distribution of cells ...

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

创新 RNA原位检测技术在 D1-Alpha 鼠伏隔核中多巴胺受体的鉴定

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in ...

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 ...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

小鼠大脑细胞核巴沙里斯中胆碱能纤维长度的立体估计

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...

Elimination of Serotonergic Neurons by Stereotaxic Injection of 5,7-Dihydroxytryptamine in the Dorsal Raphe Nuclei of Mice

Stereotaxic injection has been widely used for direct delivery of compounds or viruses to targeted brain areas in rodents. Direct targeting of serotonergic neurons in the dorsal raphe nucleus (DRN) can cause excessive bleeding and animal death, due to its location below the superior sagittal sinus (SSS). This protocol describes the generation of a DRN serotonergic neuron-lesioned mouse model (&#62;90% survival rate) with stable loss of &#62;70% 5-HT-positive cells in the DRN. The lesion is induced by stereotaxic injection of a selective serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into the DRN using an angled approach (30&#176; in the anterior/posterior direction) to avoid injury to the SSS. DRN serotonergic neuron-lesioned mice display anxiety-associated behavior alterations, which helps to confirm success of the DRN lesion surgery. This method is used here for DRN lesions, but it can also be used for other stereotaxic injections that require angular injections to avoid midline structures. This DRN serotonergic neuron-lesioned mouse model provides a valuable tool for understanding the role of serotonergic neurons in the pathogenesis of psychiatric disorders, such as generalized anxiety disorder and major depressive disorder.

This protocol describes a dorsal raphe nucleus (DRN)-lesioned mouse model (&#62;90% survival rate in experimental mice) with stable loss of dorsal raphe serotonergic neurons by stereotaxic injection of 5,7-dihydroxytryptamine into the DRN using an angled approach to prevent injury to the superior sagittal sinus.

Stereotaxic injection has been widely used for direct delivery of compounds or viruses to targeted brain areas in rodents. Direct targeting of ...

Studying Pre-formed Fibril Induced &#945;-Synuclein Accumulation in Primary Embryonic Mouse Midbrain Dopamine Neurons

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on &#945;-synuclein aggregation in neuronal cultures. Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. In this protocol, the hallmark histopathology of Parkinson&#8217;s disease, Lewy bodies (LB), is mimicked by the addition of &#945;-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media. Accumulation of endogenous phosphorylated &#945;-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing &#945;-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates. These data are quantified (e.g., counting the number of phospho-&#945;-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. PFF-induced modeling of phosphorylated &#945;-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of &#945;-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.

Here, we present a detailed protocol to study neuronal &#945;-synuclein accumulation in primary mouse dopamine neurons. Phosphorylated &#945;-synuclein aggregates in neurons are induced with pre-formed &#945;-synuclein fibrils. Automated imaging of immunofluorescently labeled cells and unbiased image analysis make this robust protocol suitable for medium-to-high throughput screening of drugs that inhibit &#945;-synuclein accumulation.

研究原发性胚胎小鼠中脑多巴胺神经元中预制纤维诱导β-合成素积累

The goal of this protocol is to establish a robust and reproducible model of &#945;-synuclein accumulation in primary dopamine neurons. Combined with ...

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased stereological counting is the standard for quantification of these cells, but it remains a laborious and time-consuming process, which may not be feasible for all projects. Here, we describe the use of an image analysis platform, which can accurately estimate the quantity of labeled cells in a pre-defined region of interest. We describe a step-by-step protocol for this method of analysis in rat brain and demonstrate it can identify a significant reduction in tyrosine hydroxylase positive neurons due to expression of mutant &#945;-synuclein in the substantia nigra. We validated this methodology by comparing with results obtained by unbiased stereology. Taken together, this method provides a time-efficient and accurate process for detecting changes in dopaminergic neuron number, and thus is suitable for efficient determination of the effect of interventions on cell survival.

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

使用自动图像分析对啮齿动物模型的亚斯坦蒂娅尼格拉多巴明神经元密度进行半定量测定

Estimation of the number of dopaminergic neurons in the substantia nigra is a key method in pre-clinical Parkinson's disease research. Currently, unbiased ...