这项工作是支持希拉和大卫恩特神经病理性疼痛研究计划研究生奖学金 (J.M.B.), 露易丝教皇生活研究员计划 (铁匠, Y.Z., 和 J.M.B.), 斯奈德-鲁滨逊基金会 Predoctoral 奖学金 (铁匠), 约翰博士 T.麦克唐纳基金会 (铁匠), 合同, 国家卫生研究院 (NIH) HHSN268201300038C, R21GM119018 和 R56NS095893 (R.G.Z.) 的赠款, 并由泰山学者项目 (山东省, 中华人民共和国) (R.G.Z.)。

1. 图像分析实验设计的思考与准备
<ol><li>预先确定合适的解剖, 细胞, 或亚型标记, 以作为标志, 以标准化的兴趣区域 (ROI) 横跨不同的样本, 例如 4 ', 6-diamidino 2-苯基吲哚 (DAPI), 膜标记, 局部荧光蛋白等.
</li>
	<li>选择一个荧光标记, 它可以将离散 puncta 划分为兴趣结构以外的背景层。 注: 本协议是为蛋白质结构 (如错误折叠蛋白聚集) 和膜结合器 (如自噬记者) 进行优化的。为了可视化蛋白质聚集, 使用了一个 RFP 标记的 n-末端片段的人 Htt 蛋白与致病 polyQ 道138重复 (RFP-hHttQ138)5。突变 Htt 蛋白在神经元特异驱动物 (如elav-GAL45、6) 下表达时形成细胞质团聚体。为了形象化 autophagosome 溶酶体的通量, actin-GAL4被用来无所不在串联 mCherry-GFP-Atg8a 记者的驱动表达。mCherry 和 GFP 阳性 puncta 表明自噬体, 当 GFP 荧光在溶酶体酸性环境中被淬火时, 通量被监测, 酸不敏感的 mCherry 一直保持到蛋白质降解7。对于比例分析, 作为结构的一致标记的单个通道可以用于分割, 或者如果适当, 可以使用两个或多个通道的投影来描绘结构, 尽管本协议没有明确描述。在这个例子中, mCherry 是用于 Atg8-positive 粒子的分割, 因为它是酸不敏感的, 并将保持在车厢贯穿整个通路通量。</li>
	<li>下载并安装开放源码图像分析平台 ImageJ 或斐济-ImageJ 的首选分布与捆绑的插件2,3。</li>
	<li>为 h 极大交互分水岭安装插件。 注: 本议定书使用斐济;ImageJ 可能需要额外的插件。Benoit Lombardot 为超临界-MPI-CBG 开发的插件可在线提供 (https://imagej.net/Interactive_Watershed)。</li>
	<li>导航到 "帮助 |更新 ", 选择" 管理更新站点 "从" ImageJ 更新程序 ", 从列表中选择" 超临界-MPI-CBG 更新站点 ", 然后关闭并重新启动斐济。</li>
</ol>2. 脑解剖和免疫荧光染色
<ol><li>
做硅胶衬里的解剖碟。
	<ol>
<li>将硅胶元件倒入烧杯中, 由制造商指示, 搅拌良好, 直至彻底混合。</li>
		<li>用吸管填充5厘米组织培养皿1/3 至半满 (大约10毫升的弹性体混合物)。允许任何气泡上升到表面, 并用纸巾轻轻地移除。把盘子盖好, 放在一层表面, 在室温下 48–72 h。</li>
	</ol>
</li>
	<li>
解剖果蝇的大脑和视觉系统, 如果需要的话。
	<ol>
<li>执行果蝇大脑解剖, 如前所述8,9。在弹性衬里的解剖皿中进行解剖, 使用弹性体底部稳定大脑以避免损伤组织或镊子。</li>
		<li>为了在解剖过程中保持叶片完好无损, 在视网膜下滑动两个钳, 轻轻地撕裂眼睛的中间, 以去除视网膜。用与椎板表面平行的镊子拔出附着在叶片上的任何视网膜组织。 注: 残留色素将在以下步骤洗涤, 通常不会干扰抗体染色和成像。</li>
		<li>稀释37% 甲醛在磷酸盐缓冲盐水 (PBS) 使最后的集中3.7% 新鲜之前使用在500µL 离心管。用固定溶液将解剖过的大脑转移到离心管, 在 RT 中孵育15分钟, 并进行轻柔的摇摆。 注: 甲醛具有天然的氧化倾向, 可产生甲酸。因此, 建议整除37% 甲醛储存, 并稀释到3.7% 新鲜之前, 每次使用。过度固定或不固定会影响组织的完整性和荧光10。</li>
		<li>洗涤3次与 PBTx (PBS 与0.4% 海卫 X-100) 为15分钟每。</li>
	</ol>
</li>
	<li>
执行免疫组化和装标本。
	<ol>
<li>如果需要抗体染色, 去除 PBTx, 添加原抗体稀释在 PBTx 与5% 正常山羊血清, 并孵化在一个整除搅拌机过夜4摄氏度。</li>
		<li>去除原抗体, 用 PBTx 洗3次, 每次15分钟。添加二次抗体稀释在 PBTx 与5% 正常山羊血清和孵育1小时在 RT 或隔夜在4摄氏度。用 PBTx 洗3次, 每人15分钟。</li>
		<li>如果需要 DAPI 染色, 去除 PBTx, 添加 DAPI 溶液 (股票溶液: 5 毫克/毫升, 稀释到工作浓度的15µg/毫升在 PBTx), 并孵化10分钟在 RT. 每15分钟洗3次。</li>
		<li>要清除组织, 请将一滴安装介质放到显微镜幻灯片的中心, 两侧有一层透明胶带的间隔。把大脑转移到安装培养基的下落上, 等到大脑变得透明时再等待。用吸管小心地取下安装介质。</li>
		<li>要装入, 请将一滴新鲜的安装介质放到幻灯片上的大脑上。小心地覆盖一个盖子玻璃避免气泡。用橡胶水泥封边, 在 RT 上风干20分钟. 进行图像采集以获得最佳效果。</li>
	</ol>
</li>
</ol>3. 图像采集
<ol><li>优化显微镜采集参数, 包括空间分辨率、目标、变焦、扫描速度和步长, 以最大程度地解决感兴趣的结构, 以便在图像分析过程中实现半自动化分割。 注意: 在对所有实验样本进行成像之前, 捕获一个样本图像并测试半自动分割 (在本协议的步骤5中) 可能很有用。这样, 用户就可以调整成像设置, 以便分析工具可以很容易地辨别出感兴趣的生物结构。</li>
	<li>
调整图像检测器控制, 捕捉最高的信噪比和最高的动态范围的标本。
	<ol>
<li>在调整设置时, 请使用查找表 (查找表格), 指示像素饱和度 (曝光过大) 或钻井 (偏移过低) (图 3B, 红色表示由高/低的查找像素饱和度)。</li>
		<li>验证增益和偏移设置适用于所有实验组, 因为实验操作可能会改变兴趣结构。 注意: 所有组都必须使用相同的设置进行量化比较。</li>
	</ol>
</li>
	<li>图像通过组织捕获所有数据。 注意: 建议在一个映像会话期间捕获所有可用数据, 并在需要时在步骤4中的 ROI 选择过程中定义更精确的区域。</li>
	<li>将图像保存为影像软件支持的文件类型, 以保留元数据 (如采集参数和空间校准)。保存图像的文件名包括实验日期、遗传学背景、荧光记者、抗体或与扫描的通道相对应的染料, 如 [实验组名称] _ [标本名] _ [实验日期] _Ch1. [荧光-目标/染料] _Ch2 [荧光靶/染料]等。
</li>
</ol>4. 斐济/ImageJ 图像导入和 ROI 选择
<ol><li>在斐济打开图像。使用 "生物格式导入选项" 对话框, 选择 "查看堆栈: Hyperstack" 并设置 "颜色模式: 灰度"。 注: 建议打开显微镜文件类型, 如空间标定等元数据可由斐济阅读。</li>
	<li>
从单个 z 平面或基于标记通道 (可用作跨样本的标准 roi) 的投影标识区域 (图 1, ROI 选择)。
	<ol>
<li>使用 "C" 滚动条查看用于捕获步骤1.1 中指定的标记的通道。</li>
		<li>使用 "Z" 滚动条在焦点平面中移动。选择单个切片或通过单击 "图像 |" 来创建多个切片的投影。栈 |Z 项目 "并设置" 开始切片 "和" 停止切片 "以包含 ROI。 注意: 将 " 投影类型" 设置为 "Z 项目" 对话框中的 "最大强度" 通常最适合强调5节中的细分结构, 尽管这可能不适用于所有应用程序。如果分析需要另一种类型的投影, 请注意保存并记录此文件, 以便在6和/或7节中提取特征。</li>
		<li>生成包含感兴趣的信道和焦点平面的新图像, 以简化图像分析协议中的以下步骤。选择 "图像 |重复 ", 设置所需的" 通道 "(c) 和" 切片 "(z), 并更改文件名以反映所选内容 (例如, [实验组名称] _ [样本名称] _ [实验日期] _ [通道] _ [z 平面])。</li>
	</ol>
</li>
	<li>使用斐济工具栏上的 "选择工具" 之一, 根据步骤4.2 中确定的选择标准手动选择标准化的 ROI。</li>
	<li>单击 "分析", 将 roi 添加到 roi 管理器中。工具 |roi 管理器 ", 然后单击" roi 管理器 "菜单上的" 添加 "。单击 "更多 |", 从 "roi 管理器" 菜单中保存 roi保存 ", 然后命名该文件以反映 ROI (例如, [实验组名称] _ [样本名称] _ [实验日期] _ [频道] _ [z 面] _ [ROI 说明])。 注: 这可以在斐济重新开放, 以便以后进行分析, 也可以应用于5.3 节中的其他渠道或图像。</li>
</ol>5. h 极大 Watershedding 的预处理与分割
<ol><li>
对用于捕获感兴趣结构的通道执行预处理。
	<ol>
<li>如果图像文件由多个通道组成, 请通过单击 "图像 |" 来分隔通道。颜色 |分割渠道 ", 以隔离的渠道利益。</li>
		<li>应用一个过滤器, 如中值过滤器, 以减少噪音或高斯模糊的平滑通过单击 "过程 |过滤器 |过滤器类型 "(例如," 中值滤波 "或" 高斯模糊 ")。<ol>
<li>在每个实验组的图像上取样不同的过滤器和过滤器参数。从每个组中的一个典型示例开始步骤 6.2, 以检查分割性能。选择便于细分利益结构的筛选器和设置。</li>
		</ol>
</li>
		<li>记录筛选器和设置。在实验组中应用这些参数。</li>
		<li>将图像的副本另存为 tiff 文件, 包括应用的筛选器以供参考。使用详细名称, 如 [实验组名称] _ [样本名称] _ [实验日期] _ [通道] _ [z 面] _ [带参数的过滤器]。</li>
	</ol>
</li>
	<li>
在步骤5.1 中生成并保存的预处理图像上, 使用交互式的 h 极大值分水岭工具对感兴趣的结构进行分割。
	<ol>
<li>利用在斐济的预处理图像, 通过点击 "超临界值" 来启动 h 极大交互分水岭工具;标签 |交互式 H_Watershed "。 注意: 这个插件首先计算分水岭, 然后允许用户通过一个即时更新的输出图像来探索局部极大值和门限选项对分割的影响。</li>
		<li>从控制面板, 调整种子动力学 (h), 强度阈值 (T), 峰值洪水 (%), 以优化分割。 注: 在4.2 节预处理前, 请始终参考原始图像, 以手动检查感兴趣结构的细分性能。</li>
		<li>当对分割结果满意时, 选择 "导出区域掩码" 并选择 "导出"。这将生成分水岭结果的二进制图像 (图 1, 分割)。</li>
		<li>记录分割参数;这些参数需要在实验组中应用以进行定量比较。保存二进制结果掩码 (如果需要参考), 并在名称 (如 [实验组名称] _ [样本名称] _ [实验日期] _ [通道] _ [z 面] _ [H_Watershed 参数] _ 中详细描述了细分参数。</li>
	</ol>
</li>
	<li>
从分水岭结果中提取出感兴趣的结构。
	<ol>
<li>在 ROI 管理器中打开步骤4.4 中保存的 roi。使用步骤5.2.4 活动中的二进制区域掩码, 从 roi 管理器中选择要应用于图像的 roi, 以将特征提取限制为标准化的 ROI。</li>
		<li>利用 ROI 在二进制分水岭掩码上活动, 选择 "分析 |在对话框中选择 "添加到 Manager" 来分析粒子以提取特征。 注意: 这将为细分期间为 ROI 管理器划分的每个结构添加一个粒子标识符。此菜单上的选项可在需要时根据大小或循环来排除特征, 以细化分析中包含的结构。</li>
		<li>通过单击 "更多 |" 来保存粒子从 ROI 管理器菜单中保存 "。确保取消选择标识符, 所有粒子都将保存在 zip 文件中。使用一个详细的名称, 如 [实验组名称] _ [样本名称] _ [实验日期] _ [通道] _ [z 面] _ [用参数过滤] _ [H_Watershed 参数] _ [结构]。</li>
	</ol>
</li>
</ol>6. 数量、面积和强度的量化和分析
<ol><li>打开生成并保存在步骤4.2 中的原始映像。滚动到用于捕获感兴趣结构的通道。 注意: 从原始图像 (预处理前) 进行强度测量非常关键, 因为在步骤5.1 中应用滤镜会更改像素值。</li>
	<li>打开步骤5.3 中从特征提取获得的粒子, 然后从 ROI 管理器中选择 "显示全部"。 注意: 此操作将将 "ROI 管理器" 上每个粒子的轮廓叠加到图像上, 并应匹配感兴趣结构的边缘 (图 1, 特征提取)。</li>
	<li>通过单击 "分析 |" 来设置所需的度量值设置测量 "。 注意: 粒子的面积、强度和密度可以通过选择 "区域" 和 "集成密度" 来测量。当选择集成密度时, 斐济将产生两个值: "集成密度", 计算为区域的乘积和平均值的灰度值和 "原始集成密度", 测量为像素值的总和。荧光蛋白在粒子中的密度被计算为除以区域的像素值的总和。步骤5.3 中生成的粒子数表明步骤4.4 中定义的组织 ROI 中的粒子数量。</li>
	<li>从 ROI 管理器菜单中选择 "度量"。只要测量是从图像软件中从斐济获得的文件中提取的, 结果就用校准单位表示。从结果窗口复制结果并粘贴到电子表格软件中进行编译和进一步计算。</li>
</ol>7. 比例量化和数据分析
<ol><li>按照步骤6.1 和 6.2, 打开感兴趣的第一个原始通道上的粒子标识符。</li>
	<li>通过单击 "分析 |" 来设置所需的度量值设置测量 "。 注: 通过选择 "平均灰度值" 可以测量每个粒子的平均强度。</li>
	<li>从 ROI 管理器中选择 "度量"。从结果窗口复制数据并粘贴到电子表格软件中。</li>
	<li>将 "C" 滚动条移至感兴趣的第二个原始通道, 此示例中的 GFP (图 4A), 然后重复步骤7.2。和7.3。 注意: 在步骤5.3 中保存的粒子将默认为生成它的通道和切片。注意确保粒子被应用到正确的感兴趣的通道上。</li>
	<li>计算电子表格软件中每个粒子的强度与第二个通道强度的比值。</li>
	<li>
使用散点图可视化和量化来自显影的比例数据, 它们反映了潜在的生物过程的个人变化。
	<ol>
<li>在图形软件中生成散点图。 注意: 每个数据点表示一个感兴趣的结构, 其中第一个荧光的强度是 "x 值", 第二个荧光的强度是从每个粒子测量的 "y 值"。</li>
		<li>为每个荧光设置浇口阈值以定义象限。</li>
	</ol>
</li>
	<li>
使用线条轮廓可视化的荧光强度跨结构的兴趣。
	<ol>
<li>选择在步骤5.3 中生成的 ROI, 并使用工具栏上的直线工具通过 roi 绘制一条直线。将行 roi 添加到 roi 管理器中。</li>
		<li>对于每个感兴趣的频道, 用行 ROI 活动, 选择 "分析 |剧情简介 "。 注意: 剧情剖面函数将生成一个直方图图和一个表的强度值沿线。复制每个通道测量的结果并粘贴到电子表格软件中, 以生成显示沿线的两个通道的图。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Erkkinen, M. G., Kim, M. -. O., Geschwind, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+Neurology+and+Epidemiology+of+the+Major+Neurodegenerative+Diseases.">Clinical Neurology and Epidemiology of the Major Neurodegenerative Diseases.</a> Cold Spring Harbor perspectives in biology. , a033118 (2017).</li><li>Schindelin, 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=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature methods. 9 (7), 676-682 (2012).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature methods. 9 (7), 671 (2012).</li><li>Mauvezin, C., Ayala, C., Braden, C. R., Kim, J., Neufeld, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+to+monitor+autophagy+in+Drosophila.">Assays to monitor autophagy in Drosophila.</a> Methods. 68 (1), 134-139 (2014).</li><li>Weiss, K. R., Kimura, Y., Lee, W. -. C. M., Littleton, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntingtin+aggregation+kinetics+and+their+pathological+role+in+a+Drosophila+Huntington's+disease+model.">Huntingtin aggregation kinetics and their pathological role in a Drosophila Huntington's disease model.</a> Genetics. 190 (2), 581-600 (2012).</li><li>Babcock, D. T., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcellular+spreading+of+huntingtin+aggregates+in+the+Drosophila+brain.">Transcellular spreading of huntingtin aggregates in the Drosophila brain.</a> Proceedings of the National Academy of Sciences. 112 (39), E5427-E5433 (2015).</li><li>DeVorkin, L., Gorski, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+autophagy+in+Drosophila+using+fluorescent+reporters+in+the+UAS-GAL4+system.">Monitoring autophagy in Drosophila using fluorescent reporters in the UAS-GAL4 system.</a> Cold Spring Harbor Protocols. 2014 (9), (2014).</li><li>Williamson, W. R., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+developing+and+adult+Drosophila+brains+and+retinae+for+live+imaging.">Preparation of developing and adult Drosophila brains and retinae for live imaging.</a> Journal of visualized experiments: JoVE. (37), (2010).</li><li>Sweeney, S. T., Hidalgo, A., de Belle, J. S., Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+adult+Drosophila+brains.">Dissection of adult Drosophila brains.</a> Cold Spring Harbor Protocols. (12), (2011).</li><li>Fox, C. H., Johnson, F. B., Whiting, J., Roller, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+fixation.">Formaldehyde fixation.</a> Journal of Histochemistry &amp; Cytochemistry. 33 (8), 845-853 (1985).</li><li>Lee, T., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+analysis+with+a+repressible+cell+marker+(MARCM)+for+Drosophila+neural+development.">Mosaic analysis with a repressible cell marker (MARCM) for Drosophila neural development.</a> Trends in neurosciences. 24 (5), 251-254 (2001).</li><li>Fischbach, K. -. F., Dittrich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+optic+lobe+of+Drosophila+melanogaster.+I.+A+Golgi+analysis+of+wild-type+structure.">The optic lobe of Drosophila melanogaster. I. A Golgi analysis of wild-type structure.</a> Cell and tissue research. 258 (3), 441-475 (1989).</li><li>Ruan, K., Zhu, Y., Li, C., Brazill, J. M., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+Drosophila+Nmnat+functions+as+a+switch+to+enhance+neuroprotection+under+stress.">Alternative splicing of Drosophila Nmnat functions as a switch to enhance neuroprotection under stress.</a> Nature Communications. 6, 10057 (2015).</li><li>Zhai, R. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NAD+synthase+NMNAT+acts+as+a+chaperone+to+protect+against+neurodegeneration.">NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration.</a> Nature. 452 (7189), 887 (2008).</li><li>Li, C., 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=Spermine+synthase+deficiency+causes+lysosomal+dysfunction+and+oxidative+stress+in+models+of+Snyder-Robinson+syndrome.">Spermine synthase deficiency causes lysosomal dysfunction and oxidative stress in models of Snyder-Robinson syndrome.</a> Nat Commun. 8 (1), 1257 (2017).</li><li>Prokop, A., Meinertzhagen, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+structure+of+synaptic+contacts+in+Drosophila.">Development and structure of synaptic contacts in Drosophila.</a> Semin Cell Dev Biol. 17 (1), 20-30 (2006).</li><li>Maday, S., Holzbaur, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagosome+biogenesis+in+primary+neurons+follows+an+ordered+and+spatially+regulated+pathway.">Autophagosome biogenesis in primary neurons follows an ordered and spatially regulated pathway.</a> Dev Cell. 30 (1), 71-85 (2014).</li><li>Mizushima, N., Yamamoto, A., Matsui, M., Yoshimori, T., Ohsumi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+analysis+of+autophagy+in+response+to+nutrient+starvation+using+transgenic+mice+expressing+a+fluorescent+autophagosome+marker.">In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker.</a> Molecular biology of the cell. 15 (3), 1101-1111 (2004).</li><li>Lee, S., Sato, Y., Nixon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+lysosomal+dysfunction+causes+cargo-specific+deficits+of+axonal+transport+leading+to+Alzheimer-like+neuritic+dystrophy.">Primary lysosomal dysfunction causes cargo-specific deficits of axonal transport leading to Alzheimer-like neuritic dystrophy.</a> Autophagy. 7 (12), 1562-1563 (2011).</li><li>Vincent, L., Soille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Watersheds+in+digital+spaces:+an+efficient+algorithm+based+on+immersion+simulations.">Watersheds in digital spaces: an efficient algorithm based on immersion simulations.</a> IEEE Transactions on Pattern Analysis &amp; Machine Intelligence. (6), 583-598 (1991).</li><li>Najman, L., Schmitt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geodesic+saliency+of+watershed+contours+and+hierarchical+segmentation.">Geodesic saliency of watershed contours and hierarchical segmentation.</a> IEEE Transactions on pattern analysis and machine intelligence. 18 (12), 1163-1173 (1996).</li><li>Shiber, A., Breuer, W., Ravid, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+quantification+and+characterization+of+intracellular+protein+aggregates+in+yeast.">Flow cytometric quantification and characterization of intracellular protein aggregates in yeast.</a> Prion. 8 (3), 276-284 (2014).</li><li>Mizushima, N., Yoshimori, T., Levine, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+mammalian+autophagy+research.">Methods in mammalian autophagy research.</a> Cell. 140 (3), 313-326 (2010).</li></ol>

作者没有什么可透露的。

此处概述的协议可用于重现性定量细胞生物学过程的荧光成像。必须仔细考虑生物环境和技术限制, 以指导实验设计。荧光标记的兴趣, 无论是免疫组织化学, 染料基础, 或基因表达, 需要区分以上背景的形态学和强度。UAS/GAL4系统广泛应用于果蝇的靶向基因表达。这里的例子使用了携带 GAL4 驱动程序的转基因线激活荧光标记蛋白的转录在无人驾驶增强器的控制下, 以调查蛋白质聚集和?...

神经退行性疾病每年影响上百万人, 发病率随着人口老龄化1而增加。尽管每种神经退行性疾病都有一个独特的病因, 错误折叠蛋白的聚集和 proteostasis 网络的崩溃是许多这些疾病的常见病理特征。阐明这些基本和相互关联的过程的中断如何导致神经元功能障碍, 细胞死亡对于理解神经退行性疾病以及指导治疗干预是至关重要的。荧光成像技术允许对神经元的这些复杂和动态过程进行调查, 并极大地促进了我们对神经细胞生物学的理解。荧光标记蛋白的分析具有挑战性, 尤其是在体内进行实验时, 由于组织高度紧凑, 细胞类型多样, 形态学异质性。人工辅助量化是负担得起和直接的, 但往往是费时的, 并受人的偏见。因此, 需要有无偏见的、可重复的和易于获取的方法来从成像研究中提取量化数据。
我们概述了一个简单和适应性的工作流程使用斐济/ImageJ, 一个强大的和自由访问的图像处理软件2,3, 从荧光成像研究中提取定量数据的实验模型变性使用果蝇。通过遵循这一协议来量化蛋白质聚集和自噬性通量-两个细胞生物学特征, 这是高度相关的神经退行性疾病病理学-我们展示了敏感性和再现这种方法。荧光标记突变体杭丁顿蛋白 (Htt) 蛋白在果蝇视神经叶中的分析揭示了蛋白质聚集的数量、大小和强度。我们在果蝇视觉系统中可视化了自噬性通量的串联荧光记者, 它根据区划环境4显示不同的发射信号。比例分析的串联记者允许从 autophagosome 的形成, 成熟和运输到降解溶酶体的自噬溶酶通量的定量和全面的看法, 并进一步强调脆弱神经退行性疾病的车厢中断。重要的是, 在这两种分析中, 我们在协议中实现了半自动阈值和分割步骤, 以最大限度地减少无意识偏差, 增加采样功率, 并提供一个标准来促进类似研究之间的比较。简单的工作流旨在使强大的斐济/ImageJ 插件 (由计算机科学家根据数学算法开发) 更容易被 neurobiologists 和生命科学社区访问。

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		<tr height="38">
			<td height="38" style="height:38px;width:347px;">SYLGARD(R) 184 Silicone Elastomer Kit</td>
			<td style="width:164px;">Dow Corning Corporation</td>
			<td style="width:243px;">PF184250</td>
			<td style="width:349px;">Dissection dish</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">Falcon 35 mm Not TC-Treated Easy-Grip Style Bacteriological Petri Dish</td>
			<td style="width:164px;">Corning</td>
			<td style="width:243px;">351008</td>
			<td style="width:349px;">Dissection dish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dumont #5 Forceps</td>
			<td style="width:164px;">Fine Science Tools</td>
			<td style="width:243px;">11251-20</td>
			<td style="width:349px;">Dissection tool</td>
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			<td height="20" style="height:20px;width:347px;">Sodium Chloride</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">S3014</td>
			<td style="width:349px;">PBS solution</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Sodium Phosphate Dibasic&#160;</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">S5136</td>
			<td style="width:349px;">PBS solution</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Potassium Phosphate Monobasic</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">P5655</td>
			<td style="width:349px;">PBS solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Triton&#160;X-100</td>
			<td style="width:164px;">Sigma</td>
			<td style="width:243px;">T9284</td>
			<td style="width:349px;">Washing and antibody incubaton solution.&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:347px;">37% Formaldehyde</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">10790-710</td>
			<td style="width:349px;">Fixation</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Disposable Microcentrifuge Tubes (0.5mL, blue)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">89000-022</td>
			<td style="width:349px;">Fixation, washing, and antibody incubaton.&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Plain and Frosted Micro Slides (25&#215;75mm)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">48312-004</td>
			<td style="width:349px;">Slides for confocal imaging</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Micro Cover Glasses, rectangular (22&#215;40mm)</td>
			<td style="width:164px;">VWR</td>
			<td style="width:243px;">48393-172</td>
			<td style="width:349px;">Slides for confocal imaging</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Rubber Cement</td>
			<td style="width:164px;">Slime</td>
			<td style="width:243px;">1051-A</td>
			<td style="width:349px;">Mounting</td>
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		<tr height="38">
			<td height="38" style="height:38px;width:347px;">VECTASHIELD Antifade Mounting Medium</td>
			<td style="width:164px;">Vector Laboratories, Inc.</td>
			<td style="width:243px;">H-1000</td>
			<td style="width:349px;">Mounting</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Scotch Magic 810 Invisible Tape (19mm&#215;25.4m)</td>
			<td style="width:164px;">3M Company</td>
			<td style="width:243px;">810</td>
			<td style="width:349px;">Mounting</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">Normal Goat Serum</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:243px;">PCN5000</td>
			<td style="width:349px;">Antibody incubaton</td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:347px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)&#160;</td>
			<td style="width:164px;">Thermo Fisher Scientific</td>
			<td style="width:243px;">D1306</td>
			<td style="width:349px;">Nucleic acid staining. Dissolve in deionized water to make a 5 mg/mL stock solution and store at -80&#176;C. Dilute to a working concentration of 10-20 &#956;g/mL in PBTx.</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:347px;">3.5X-90X Stereo Zoom Inspection Industrial Microscope</td>
			<td style="width:164px;">AmScope</td>
			<td style="width:243px;">SM-1BNZ</td>
			<td style="width:349px;">Dissection scope. Equipped with 6W LED Dual Gooseneck Illuminator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">ImageJ/Fiji</td>
			<td style="width:164px;">NIH</td>
			<td style="width:243px;">v1.51u</td>
			<td style="width:349px;">With SCF_MPI_CBG plugins (version 1.1.2)</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:347px;">FV1000-IX81 Confocal-laser Scanning Microscope</td>
			<td style="width:164px;">Olympus</td>
			<td style="width:243px;"></td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Recombinant Construct</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td style="width:243px;">BL37749</td>
			<td style="width:349px;"></td>
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quantitative cell biology of neurodegeneration in drosophila through unbiased analysis of fluorescently tagged proteins using imagej

随着神经退行性疾病患病率的上升, 了解导致神经元功能障碍和丧失的潜在病理生理学越来越重要。基于荧光的成像工具和技术能够对亚细胞神经生物学过程进行空前的分析, 但仍然需要有无偏见、可重现和易于获取的方法来从成像研究中提取量化数据。.我们开发了一个简单而适应的工作流程, 从荧光成像研究中提取定量数据, 利用果蝇模型变性。具体来说, 我们描述了一个易于跟踪, 半自动化的方法, 使用斐济/ImageJ 分析两个细胞过程: 首先, 我们用荧光标记突变体量化果蝇光学叶中蛋白质的总含量和剖面。杭丁顿蛋白蛋白;第二, 我们评估果蝇视觉系统的自噬溶酶通量, 并以比例为基础, 对一个串联荧光报告器进行定量化。重要的是, 这里概述的协议包括一个半自动的分割步骤, 以确保所有的荧光结构进行分析, 以尽量减少选择偏差, 并增加微妙比较的分辨率。这种方法可以推广到分析其他细胞生物学结构和过程中涉及变性, 如蛋白质 puncta (应力颗粒和突触复合物), 以及膜约束的车厢 (线粒体和膜贩运囊泡)。该方法为图像分析和量化提供了一个标准化的、可适应的参考点, 并能促进整个领域的可靠性和重现性, 最终增强了对变性的机械理解。

果蝇荧光标记突变 Htt 集的数量、面积和强度的量化
为了研究亨廷顿氏病果蝇模型的中枢神经系统中的错误折叠蛋白聚集, 用 RFP 标记的突变体 Htt 非病理 (UAS-RFP-hHttQ15) 或病理扩张 (UAS-RFP-hHttQ138) 和膜 GFP (UAS-mCD8::GFP)11是共同表达下一个泛神经元驱动elav-GAL4。根据《议定书?...

Watch this Scientific Journal Video about Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ at JoVE.com

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

我们开发了一个简单且适应性强的工作流, 从荧光成像的细胞生物学研究中提取定量数据自噬性果蝇模型中中枢神经系统蛋白质聚集和通量变性.

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

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Research

JoVE Journal

Neuroscience

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

9.0K 次观看.  University of Miami Miller School of Medicine. 我们开发了一个简单且适应性强的工作流, 从荧光成像的细胞生物学研究中提取定量数据自噬性果蝇模型中中枢神经系统蛋白质聚集和通量变性.

视频: Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3. Specifically, it affects the ability of neurons to receive inputs from other cells2 and contributes to the propagation of action potentials4,5. The morphology of the neurites also affects how information is processed. The diversity of dendrite morphologies facilitate local and long range signaling and allow individual neurons or groups of neurons to carry out specialized functions within the neuronal network6,7. Alterations in dendrite morphology, including fragmentation of dendrites and changes in branching patterns, have been observed in a number of disease states, including Alzheimer's disease8, schizophrenia9,10, and mental retardation11. The ability to both understand the factors that shape dendrite morphologies and to identify changes in dendrite morphologies is essential in the understanding of nervous system function and dysfunction.
Neurite morphology is often analyzed by Sholl analysis and by counting the number of neurites and the number of branch tips. This analysis is generally applied to dendrites, but it can also be applied to axons. Performing this analysis by hand is both time consuming and inevitably introduces variability due to experimenter bias and inconsistency. The Bonfire program is a semi-automated approach to the analysis of dendrite and axon morphology that builds upon available open-source morphological analysis tools. Our program enables the detection of local changes in dendrite and axon branching behaviors by performing Sholl analysis on subregions of the neuritic arbor. For example, Sholl analysis is performed on both the neuron as a whole as well as on each subset of processes (primary, secondary, terminal, root, etc.) Dendrite and axon patterning is influenced by a number of intracellular and extracellular factors, many acting locally. Thus, the resulting arbor morphology is a result of specific processes acting on specific neurites, making it necessary to perform morphological analysis on a smaller scale in order to observe these local variations12.
The Bonfire program requires the use of two open-source analysis tools, the NeuronJ plugin to ImageJ and NeuronStudio. Neurons are traced in ImageJ, and NeuronStudio is used to define the connectivity between neurites. Bonfire contains a number of custom scripts written in MATLAB (MathWorks) that are used to convert the data into the appropriate format for further analysis, check for user errors, and ultimately perform Sholl analysis. Finally, data are exported into Excel for statistical analysis. A flow chart of the Bonfire program is shown in Figure 1.

We have developed a computer program to analyze neuronal morphology. In combination with two existing open source analysis tools, our program performs Sholl analysis and determines the number of neurites, branch points, and neurite tips. The analyses are performed so that local changes in neurite morphology can be observed.

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

科研

神经科学

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

含量的运动能力，学习和记忆的赤字果蝇</em神经退行性疾病>模型

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences. For instance, addiction to nicotine via tobacco smoking is a leading cause of premature death worldwide (World Health Organization) and is likely caused by an alteration of ion channel distribution in the brain1. Chronic nicotine exposure in both rodents and humans results in increased numbers of nicotinic acetylcholine receptors (nAChRs) in brain tissue1-3. Similarly, alterations in the glutamatergic GluN1 or GluA1 channels have been implicated in triggering sensitization to other addictive drugs such as cocaine, amphetamines and opiates4-6.

Consequently, the ability to map and quantify distribution and expression patterns of specific ion channels is critically important to understanding the mechanisms of addiction. The study of brain region-specific effects of individual drugs was advanced by the advent of techniques such as radioactive ligands. However, the low spatial resolution of radioactive ligand binding prevents the ability to quantify ligand-gated ion channels in specific subtypes of neurons. 

Genetically encoded fluorescent reporters, such as green fluorescent protein (GFP) and its many color variants, have revolutionized the field of biology7.By genetically tagging a fluorescent reporter to an endogenous protein one can visualize proteins in vivo7-10. One advantage of fluorescently tagging proteins with a probe is the elimination of antibody use, which have issues of nonspecificity and accessibility to the target protein. We have used this strategy to fluorescently label nAChRs, which enabled the study of receptor assembly using F&ouml;rster Resonance Energy Transfer (FRET) in transfected cultured cells11.More recently, we have used the knock-in approach to engineer mice with yellow fluorescent protein tagged &alpha;4 nAChR subunits (&alpha;4YFP), enabling precise quantification of the receptor ex vivo at submicrometer resolution in CNS neurons via spectral confocal microscopy12. The targeted fluorescent knock-in mutation is incorporated in the endogenous locus and under control of its native promoter, producing normal levels of expression and regulation of the receptor when compared to untagged receptors in wildtype mice. This knock-in approach can be extended to fluorescently tag other ion channels and offers a powerful approach of visualizing and quantifying receptors in the CNS.

In this paper we describe a methodology to quantify changes in nAChR expression in specific CNS neurons after exposure to chronic nicotine. Our methods include mini-osmotic pump implantation, intracardiac perfusion fixation, imaging and analysis of fluorescently tagged nicotinic receptor subunits from &alpha;4YFP knock-in mice (Fig. 1). We have optimized the fixation technique to minimize autofluorescence from fixed brain tissue.We describe in detail our imaging methodology using a spectral confocal microscope in conjunction with a linear spectral unmixing algorithm to subtract autofluoresent signal in order to accurately obtain &alpha;4YFP fluorescence signal. Finally, we show results of chronic nicotine-induced upregulation of &alpha;4YFP receptors in the medial perforant path of the hippocampus.

We have developed a novel technique of quantifying nicotinic acetylcholine receptor changes within subcellular regions of specific subtypes of CNS neurons to better understand the mechanisms of nicotine addiction by using a combination of approaches including fluorescent protein tagging of the receptor using the knock-in approach and spectral confocal imaging.

光谱共焦成像的荧光标记的尼古丁受体敲在慢性尼古丁政府小鼠

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

Quantitative Analysis of Climbing Defects in a Drosophila Model of Neurodegenerative Disorders

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and thus current therapeutic treatment strategies are not curative. Through the use of whole exome sequencing, an increasing number of genes have been identified to play a role in human locomotion. Despite identifying these genes, it is not known how these genes are crucial to normal locomotive functioning. Therefore, a reliable assay, which utilizes model organisms to elucidate the role of these genes in order to identify novel targets of therapeutic interest, is needed more than ever. We have designed a sensitized version of the negative geotaxis assay that allows for the detection of milder defects earlier and has the ability to evaluate these defects over time. The assay is performed in a glass graduated cylinder, which is sealed with a wax barrier film. By increasing the threshold distance to be climbed to 17.5 cm and increasing the experiment duration to 2 min&#160;we have observed a greater sensitivity in detecting mild mobility dysfunctions. The assay is cost effective and does not require extensive training to obtain highly reproducible results. This makes it an excellent technique for screening candidate drugs in Drosophila mutants with locomotion defects.

We present an optimized inexpensive and reliable negative geotaxis assay in Drosophila melanogaster as a model for neurodegenerative disorders. Being more sensitive to mild locomotor defects, this assay will help screen for potential genetic interactions and drug targets.

攀登缺陷的神经退行性疾病的果蝇模型定量分析

Locomotive defects resulting from neurodegenerative disorders can be a late onset symptom of disease, following years of subclinical degeneration, and ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Mass Histology to Quantify Neurodegeneration in Drosophila

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health worldwide. Although mammalian models have provided important insights into the underlying mechanisms of pathogenicity, the complexity of mammalian systems together with their high costs are limiting their use. Therefore, the simple but well-established Drosophila model-system&#160;provides an alternative for investigating the molecular pathways that are affected in these diseases. Besides behavioral deficits, neurodegenerative diseases are characterized by histological phenotypes such as neuronal death and axonopathy. To quantify neuronal degeneration and to determine how it is affected by genetic and environmental factors, we use a histological approach that is based on measuring the vacuoles in adult fly brains. To minimize the effects of systematic error and to directly compare sections from control and experimental flies in one preparation, we use the &#39;collar&#39; method for paraffin sections. Neurodegeneration is then assessed by measuring the size and/or number of vacuoles that have developed in the fly brain. This can either be done by focusing on a specific region of interest or by analyzing the entire brain by obtaining serial sections that span the complete head. Therefore, this method allows one to measure not only severe degeneration but also relatively mild phenotypes that are only detectable in a few sections, as occurs during normal aging.

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila is widely used as a model system to study neurodegeneration. This protocol describes a method by which degeneration, as determined by vacuole formation in the brain, can be quantified. It also minimizes effects due to the experimental procedure by processing and sectioning control and experimental flies as one sample.

大众组织学量化在神经退行性变<em&gt;果蝇</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive crawling behavior.

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

嗜德维人神经元树突状树突状复杂度的定量分析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

An Unbiased Approach of Sampling TEM Sections in Neuroscience

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative studies of the changes in densities and distributions of such features, an unbiased sampling protocol is vital for reliable results. Here, we present a workflow for the image acquisition of brain samples. The workflow allows systematic uniform random sampling within a defined brain region, and the images can be analyzed using a disector. This technique is much faster than extensive examination of serial sections but still presents a feasible approach to estimate the densities and distributions of ultrastructure features. Before embedding, stained vibratome sections were used as a reference to identify the brain region under investigation, which helped speed up the overall specimen preparation process. This approach was used for comparative studies investigating the effect of an enriched-housing environment on several ultrastructural parameters in the mouse brain. Based on the successful use of the workflow, we adapted it for the purpose of elemental analysis of brain samples. We optimized the protocol in terms of the time of user-interaction. Automating all the time-consuming steps by compiling a script for the open source software SerialEM helps the user to focus on the main work of acquiring the elemental maps. As in the original workflow, we paid attention to the unbiased sampling approach to guarantee reliable results.

We introduce a novel workflow for electron microscopy investigations of brain tissue. The method allows the user to examine neuronal features in an unbiased fashion. For elemental analysis, we also present a script that automatizes most of the workflow for randomized sampling.

神经科学中透射电镜切片采样的一种无偏方法

Investigations of the ultrastructural features of neurons and their synapses are only possible with electron microscopy. Especially for comparative ...