我们感谢台湾科技部 MOST105-2628-038-005-MY3 (1-3) 提供的赠款, 以及烟草产品的健康和福利附加费--MOHW106-TDU-B-212-144001 至 Y C。

台北医科大学动物保育和使用委员会 (IACUC) 批准了所有动物实验的协议和方法 (协议编号: LAC-2013-0278;LAC-2015-0397)
1. 原代神经元培养
<ol><li>准备在文化之前
	<ol>
<li>在文化的前一天, 用聚 d-赖氨酸 (表 1) 来涂上培养皿。对于一个6的好菜, 加入1.5 毫升聚 d-赖氨酸 (0.05 毫克/毫升) 到每一个井。对于活成像, 培养细胞在一个12毫米 x 12 毫米片在一个标准被涂的 6-井板材。</li>
		<li>取出/回收聚 d-赖氨酸, 用2毫升 ddH2O 来清洗两次碗碟。</li>
		<li>准备手术工具: 一对微解剖剪刀, 弯曲尖钳, 和标准直尖钳。在手术中对手术工具进行消毒, 并在75% 乙醇中保存。</li>
		<li>用20毫升洗涤介质 (表 1) 填充 10 cm 培养皿, 并保持冰。</li>
		<li>用15毫升洗涤介质填充15毫升的试管, 并将管子放在冰上。</li>
	</ol></li>
	<li>不同脑区的脑组织分离
	<ol>
<li>牺牲16-19 天的老怀孕雌性小鼠与标准的 euthanization 协议, 放置在一个非通风笼鼠, 然后陶醉与 CO2。E15.5 ~ E16.5 胚胎被推荐用于下丘脑神经元培养和 E16.5 ~ E17.5 更适合海马和皮层神经元培养。</li>
		<li>用75% 乙醇消毒平台、解剖显微镜和手术工具, 避免污染。</li>
		<li>在脐带区域 (暗红色区域、图 1A、1B虚线) 周围剪切, 以便在不损坏幼崽的情况下很容易地隔离它们 (图 1C, 1D)。</li>
		<li>用一对剪刀 (图 1E, F) 卸下每个 pup 的头部部分, 然后使用眼睛区域的细尖直钳 (图 1G) 将头部部分固定到位。使用另一条细尖的弯曲钳, 从两侧取出外皮和颅骨, 并将其从前向背方向剥离 (图 1H, 黑色箭头指向方向)。大脑应该被隔离, 不受任何伤害 (图 1I)。 注意: 必须保持大脑的完整性, 以确保在隔离大脑的不同区域时的准确性。</li>
		<li>保持隔离的大脑在一个干净的培养皿与冰冷的洗涤介质, 以下步骤。</li>
		<li>翻转大脑, 保持腹侧向上。下丘脑是大脑中间的圆形结构 (图 1J, 箭头指向下丘脑区域)。用细尖的弯曲钳将下丘脑组织分离。小心地除去脑膜 (有黄红色的颜色)。 注意: 完全切除脑膜是避免成纤维细胞污染的关键步骤。</li>
		<li>用锋利的镊子抓住嗅叶, 用弯曲钳剥去整个大脑周围的薄脑膜 (图 1K, L)。</li>
		<li>海马是一种香蕉状的形状, 嵌在皮层下部。翻转到皮层的下侧, 将海马体从皮层中分离出来, 用弯曲钳 (图 1M, N,和O) 将其拉到一侧。一定要清除脑组织周围所有的脑膜。</li>
		<li>当大脑的所有区域都被隔离后, 在钳夹 (步骤 1.1.5) 的帮助下, 在含有冰冷洗涤介质的15毫升管中切下这些组织。 注: 脑组织可保存在冰冷的洗涤介质中2-3 小时。</li>
	</ol></li>
	<li>组织消化、电镀和培养
	<ol>
<li>通过将组织包含15毫升管的2-3 次反转, 冲洗组织, 然后保持导管平直, 使组织安定下来 (1-3 分钟)。</li>
		<li>使用与真空系统连接的玻璃巴斯德吸管移除上部介质。要清洗组织, 加入15毫升冰冷洗涤介质和倒置管2-3 次。在下一步之前重复洗涤步骤3次。 注意: 在使用真空系统去除上部介质时, 要小心底部的组织。</li>
		<li>最后洗涤后, 在1毫升吸管的帮助下去除洗涤介质。</li>
		<li>用木瓜蛋白酶-胰蛋白酶消化缓冲液孵育组织 (表 1) 在37° c 的水浴中, 7-14 分钟摇动管子, 确保所有的组织都正确地接触到消化缓冲液。 注: 孵化时间和消化缓冲液体积可根据组织大小进行调整。对于从6-8 只幼崽中采集的下丘脑组织, 推荐300µL 木瓜蛋白酶-胰蛋白酶消化缓冲液和7分钟孵育时间。更多的组织将需要更多的消化缓冲。</li>
		<li>通过添加1体积的胎牛血清中和酶的活性。在室温下摇动试管3-5 次。</li>
		<li>把管子放在机架上, 等待1-2 分钟, 使组织安定下来;小心地用1毫升吸管移除上清液。</li>
		<li>添加6毫升电镀介质 (表 1) 到试管中, 并吸管的组织上下50次, 用5毫升吸管轻轻 (为 6-井板, 1.5 毫升电镀介质, 每井推荐)。大多数细胞在重复移后会游离成单个细胞。 注意: 在执行此步骤时, 尽量避免气泡的形成。大多数实验室使用火烧玻璃牧场吸管, 以游离组织。一个5毫升吸管与一个狭窄的尖端可以是一个很好的替代这一步。</li>
		<li>将一根管子放在机架上, 等待1-2 分钟, 让这些矮胖的非离解组织安定下来。将含有离解细胞的上相带到新的管中, 用电镀介质稀释 (5x 体积电镀介质, 每1x 体积的离解细胞。例如, 添加5毫升电镀培养基为1毫升离解细胞)。</li>
		<li>使用例计算细胞密度, 并将所需数量的细胞加入到以多聚-d-赖氨酸为涂层的培养板中, 如步骤1.1.1。我们建议 2-4 x 105细胞每井为6井 dish/35 mm 盘推荐用于神经元成像研究。蛋白质收集和分析需要更高的密度。 注: 不要添加太多的电镀介质, 1-1. 5 毫升电镀介质在一个 6-好的菜是足够的附件。过量的培养基将延长适当的细胞附着所需的时间。</li>
		<li>等待2-3 小时, 在显微镜下检查种子神经元。神经元连接正常时会开始神经炎。</li>
		<li>取出电镀介质, 并将2毫升温热洗涤介质 (37 ° c) 放入盘中洗去电镀介质。重复此步骤两次。</li>
		<li>将2毫升完整培养基 (表 1) 添加到6井盘中的每个井中。</li>
		<li>每3-4 天更换一半培养基。每一次准备完整的培养基。从不同的小鼠脑区培养的神经细胞将有不同的形态学和特征 (图 2)。</li>
	</ol></li>
</ol>2. 脂质体系统将质粒 DNA 转染为原代神经元
注意: 对于神经元转染, 推荐使用无内毒素质粒 dna 纯化试剂盒 (材料表) 进行 dna 制备。另外的乙醇沉淀可以去除过量的溶剂, 提高 DNA 浓度。
<ol><li>
以脂质体为基础的 DNA 转染原代神经元。 注: 转染时间取决于实验所需的成熟状态。下丘脑神经元转染在 div 7-10 (div, 天体外)。<ol>
<li>dna: 脂质体混合物制备: 稀释 GFP-GLUT4 质粒 DNA16 (2 µg) 在含有300µL 低血清培养基的离心管内。在300µL 低血清培养基中, 用2µL 脂质体制备另一离心管, 在室温下孵育5分钟。</li>
		<li>将两根管子的内容结合起来, 在室温下孵育20-30 分钟。</li>
		<li>从培养皿中去除神经元培养基。在每个井中加入600µL DNA 脂质体混合物并在37° c 下孵育4-6 小时。</li>
		<li>4-6 小时后, 除去 DNA 脂质体混合物, 加入2毫升培养基。</li>
		<li>荧光信号, 如单独的 gfp 和 GLUT4 蛋白与 gfp 标签 (图 3和图 4) 共轭, 可以在18-72 小时后观察到。</li>
	</ol>
</li>
</ol>3. 实时图像记录
<ol><li>
为实时成像准备单元格
	<ol>
<li>CCR5-下丘脑神经细胞表达绿色荧光蛋白标记葡萄糖转运体 4 (GFP-GLUT4) 在 #1 上. 5 (或0.17mm 厚度) 玻璃片, 已在6井阶梯1.1.1 前涂有聚 d-赖氨酸板.</li>
		<li>小心地将片与神经元结合使用镊子, 并小心地将它放在玻璃幻灯片上。</li>
		<li>去除多余的介质/液体与微妙的任务抹布。把抹布折两次, 小心地放在片上。轻轻按下抹布不移动片。 注意: 不要用力按压玻璃片。这一步的目的是要确保片不移动/漂移在图像采集过程中引起的浮力。</li>
		<li>在反褶积显微镜舞台上放置片和滑动, 片朝下, 并妥善保护。此步骤是为了确保幻灯片位置保持不变, 以便用户可以在以后跟踪同一组目标单元格。</li>
		<li>用 60 x/1.42 NA 油浸泡物镜观察 CCR5--下丘脑神经元细胞。</li>
		<li>用 CCL5 (10 ng/毫升) 或胰岛素 (10 U/毫升) 将所选细胞样品治疗一分钟. 在片边缘添加1.5 µL 稀释的胰岛素。</li>
		<li>立即可视化并记录所选的单元格样本。节目录像软件录制30分钟。</li>
	</ol>
</li>
	<li>
反褶积显微镜与分析 注: 本协议的这一部分要求使用反褶积显微镜和专门的软件进行分析。<ol>
<li>打开成像系统的电源, 允许显微镜阶段正确初始化, 然后打开 LED 光源。</li>
		<li>在 60x 1.42 NA 物镜上加入浸入式油 (37 ° c 的活样品的折射率 1.520)。将样品幻灯片放在显微镜上, 片朝向物镜, 并正确地保护滑块。</li>
		<li>使用亮场或荧光照明来识别靶细胞。调整焦点, 直到目标细胞能被清晰地观察到。不要将物镜移动到片区域外, 以免出现不必要的划痕。</li>
		<li>用 GFP 信号识别绿色荧光蛋白共轭葡萄糖转运体 4 (GFP-GLUT4)。识别所需的目标单元以获取图像。所选的目标单元格位置可以被记住以备将来参考 (图 4)。</li>
		<li>在每个目标单元上设置适当的实验参数 (包括像素数、激发波长、传输百分比、曝光时间、叠加厚度、时间间隔和总成像时间)。对于这个实验, 图像像素数被设置为 512 x 512 (它可以设置在 1024 x 1024 的高分辨率) GFP 信号。曝光时间设置在0.025 到0.05 秒之间, 每 5 min. 次要横向 x、y 和 z 调整可以由推荐的软件 (材料表) 控制。</li>
		<li>将曝光参数设置为大约2000到3000计数, 以达到最大像素强度。为了使荧光漂白最小化, 尽量减少励磁光传输的百分比, 同时保持曝光时间少于1秒. 对每个额外的荧光通道和每个感兴趣的区域重复这些步骤。</li>
		<li>设置每个目标单元格上 Z 堆栈的上限和下限。这可以通过移动显微镜阶段, 直到目标细胞的顶部和底部都有点不集中。用户可以通过设置上下限和下限之间的图像数来调整 Z 轴的分辨率 (可以通过设置每个图像之间的距离来完成)。</li>
		<li>图像堆栈积, 后来分析了各自的软件的帮助-速度从尔在这种情况下。</li>
	</ol>
</li>
</ol>

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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=CCL5/RANTES+contributes+to+hypothalamic+insulin+signaling+for+systemic+insulin+responsiveness+through+CCR5.">CCL5/RANTES contributes to hypothalamic insulin signaling for systemic insulin responsiveness through CCR5.</a> Sci Rep. 6, 37659 (2016).</li></ol>

作者没有什么可透露的。

监测活细胞, 在 CCL5 或胰岛素刺激的能力, 是至关重要的研究 CCL5 或胰岛素对 GLUT4 运动的快速影响。事实上, 它让我们能够直观地看到在胰岛素刺激下, CCR5 和下丘脑神经元之间的显著差异.-- 。在胰岛素刺激后的不同时间点, 我们进行了内源性 GLUT4 蛋白的表面标记和 CCR5--下丘脑神经元的研究17。细胞表面蛋白的标记要求具有低背景的高特异性抗体。此外, 表面荧光量化也可能具有挑战性和耗时。因此, 时间推移记录让我们可以确定, CCL5 或胰岛素的影响是一个真正的生理变化的基础上的实验条件, 而不是一个统计的变化。结合内源性 GLUT4 的表面标记, 我们提供了有力的证据和实验证明 CCL5 和 CCR5 参与 GLUT4 易位和胰岛素信号。
在现代细胞生物学和分子生物学研究中, 许多实验都需要使用荧光显微镜。这项技术使科学家能够可视化蛋白质和/或细胞器之间的空间关系, 除了运动方向和速度、刺激效应、形态学变化和蛋白质贩运。然而, 这种技术仍然有其局限性: 当荧光兴奋时, 目标蛋白 (或区域) 发出的信号会被背景荧光所淹没。因此, 荧光图像可能会出现模糊的预期信号深埋在背景信号。这种现象在膜结合蛋白的观察中尤为明显。
全内反射荧光显微镜 (显微镜) 是为了克服这一困难而开发的。它允许科学家在不影响背景荧光的情况下, 可视化选定的表面结合荧光的激发。它使科学家能够有选择地描述特征和事件在非常稀薄的表面区域例如血浆膜。反褶积显微镜是一种计算密集型的图像处理技术, 在近几年技术进步的帮助下得以实现。它经常被用于提高数字荧光图像的分辨率。如前所述, 当荧光被任何类型的光照 (如激光或 LED) 所激发时, 所有的荧光都会发出光信号, 不管它们是否在焦点上, 因此图像总是会出现模糊。这种模糊是由一种叫做 "点扩散函数" (聚砜) 的现象引起的, 因为来自一个小的荧光源 (亮点) 的光会进一步扩散, 成为焦点 (模糊)。原则上, 这个事件将产生一个像沙漏状的荧光信号, 一个荧光图像可以由无数这样的光信号组成。反褶积过程可以将所有的荧光信号重新分配到原来的亮点形式, 消除大部分的外聚焦光, 以提高图像对比度。
近年来, 反褶积算法产生的图像具有可比分辨率的共焦显微镜。此外, 与显微镜相比, 它可以防止在有限的励磁区域检测出对焦模糊, 因此, 宽场显微镜允许检测所有的光信号, 并通过反褶积过程将其重新回源。因此, 在实践中, 反褶积显微镜不仅成为一种更有效的图像采集方法, 而且与 TIRF 显微镜相比, 更具成本效益的方法。

2型糖尿病 (T2DM) 是一个全球性的健康危机。T2DM 的特点是胰岛素信号障碍和慢性炎症的周围组织。下丘脑是调节身体能量稳态、食欲和昼夜节律的控制中心。最重要的是, 下丘脑还介导胰岛素信号应答调节全身代谢1,2,3,4,5。阻断下丘脑胰岛素信号通路可诱发胰岛素抵抗6,7。下丘脑协调细胞能量状态和激素的分泌, 例如胰岛素和脂肪 (例如, 瘦素) 从周围的组织, 调控系统的葡萄糖新陈代谢, 胰岛素反应和摄食。胰岛素结合到胰岛素受体激活胰岛素受体基质 (IRS) 蛋白, 然后激活胰岛素下游信号分子, 如 PI3K (醇 3-激酶) 和 AKT (蛋白激酶 B (PKB/AKT)), 以诱导 GLUT4膜易位。在胰岛素反应中, 神经元不是摄取葡萄糖的主要目标;然而, 在下丘脑弓状核 (弧) 区已发现了显著的 GLUT4 表达水平。因此, GLUT4 在下丘脑神经元的调节可能在脑-外周轴的胰岛素信号传导中起重要作用。
许多研究表明, 慢性炎症和炎症性趋化因子在下丘脑也起着重要的作用, 在糖尿病和肥胖的发展, 和抑制下丘脑炎症可以逆转饮食诱导胰岛素抵抗8,9,10. 此外, chemokine-CCL5 (c-c 主题配体 5, 也称为 RANTES, 调节激活-正常 t 细胞表达和分泌) 及其受体 CCR5 水平也与发展的 T2DM11,12。CCL5 和 CCR5 在胰岛素功能和葡萄糖代谢中的作用仍不清楚。一项研究报告, CCR5 缺乏保护小鼠的肥胖引起的炎症, 巨噬细胞的招募, 和胰岛素抵抗11;相比之下, 另一项研究报告说, CCR5 缺乏会损害全身葡萄糖耐受, 以及脂肪和肌肉胰岛素信号12。CCL5 被发现可以增加 T 细胞的葡萄糖摄取量, 并通过其对下丘脑的作用来减少食物摄入13,14, 但是, 作用机制和所涉及的受体还有待确定。
研究外周组织炎症对下丘脑神经元胰岛素功能影响的细胞机制是困难的。这是由于细胞异质性和神经元回路的反馈规则。因此, 一个体外细胞培养模型提供了一个干净的模型来研究趋化因子对下丘脑胰岛素信号调节的影响。虽然有许多建立永生化的下丘脑神经细胞线为研究用途, 这些细胞线表达了不同的标记, 因此, 代表不同的类型下丘脑神经元15。即使初级下丘脑的文化很难维持, 他们可以提供最现实的反应下丘脑神经元的胰岛素刺激, 也可以避免潜在的未知影响, 发挥作用时, 保持细胞长期在培养基中有人工生长因子。
在此, 我们利用 C57BL/6 野生 (小鼠) 和 CCR5 击倒 (CCR5-/-) 小鼠的下丘脑神经元, 并染两种类型的细胞 GFP-GLUT4 构造。为了研究 CCR5 对胰岛素介导的 GLUT4 膜贩运的贡献, GFP-GLUT4 转染的神经元进行胰岛素或重组 CCL5 治疗。然后, 我们用反褶积显微镜表征了 GFP-GLUT4 在下丘脑神经元的细胞膜上的运动。

<table><tbody><tr><td>DeltaVision deconvolution microscope</td><td>Applied Precision Inc./(GE Healthcare Life Science)&amp;nbsp;</td><td></td><td>equipped with 60x/1.42 NA oil immersion objective lens; used for taking live cell images</td></tr><tr><td>SoftWorX application software</td><td>Applied Precision Inc.</td><td></td><td>used to control microscope and camera</td></tr><tr><td>VoloCITY software</td><td>PerkinElmer</td><td></td><td>used to analyze the images</td></tr><tr><td>Insulin</td><td>Actrapid, Denmark</td><td></td><td></td></tr><tr><td>Liposome</td><td>Invitrogen</td><td>11668019</td><td>Lipofectamine&amp;reg; 2000, used for plasmid DNA transfection</td></tr><tr><td>GFP control plasmid DNA</td><td></td><td></td><td>provided by Professor Samuel Cushman</td></tr><tr><td>GFP-GLUT4 plasmid DNA</td><td></td><td></td><td>provided by Professor Samuel Cushman</td></tr><tr><td>Anti-mouse Alex-488</td><td>Invitrogen</td><td>#A-11001</td><td>Secondary anitbody</td></tr><tr><td>Anti-rabbit IgG -Alex-568</td><td>Invitrogen</td><td>#A-11036</td><td>Secondary anitbody</td></tr><tr><td>CCL5/RANTES</td><td>R&amp;amp;D Systems</td><td>478-MR-025</td><td>10ng/ml</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark</td><td>#FL42572A</td><td>0.17mm thickness</td></tr><tr><td>12 mm Microscope coverglass</td><td>Deckglaser</td><td>#41001112</td><td>used for immmunstaining study</td></tr><tr><td>micro-dissecting scissors</td><td>Klappenecker</td><td></td><td>Surgical tool</td></tr><tr><td>curved-tipped forceps</td><td>Ideal-tek</td><td></td><td>Surgical tool</td></tr><tr><td>standard straight-tipped forceps</td><td>Ideal-tek</td><td></td><td>Surgical tool</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>QIAGEN</td><td>12362</td><td>Purify Endotoxin free plasmid DNA</td></tr><tr><td>5 ml pipette</td><td>Corning costar&amp;nbsp;</td><td>4487</td><td>dissociate brain tissue</td></tr></tbody></table>

live images of glut4 protein trafficking in mouse primary hypothalamic neurons using deconvolution microscopy

2型糖尿病 (T2DM) 是一种全球性的健康危机, 其特点是胰岛素信号障碍和周围组织的慢性炎症。中枢神经系统 (CNS) 下丘脑是能量和胰岛素信号应答调节的控制中心。周围组织的慢性炎症和某些趋化因子 (如 CCL5、TNFα和 IL-6) 的失衡会导致糖尿病和肥胖。然而, 连接趋化因子和下丘脑胰岛素信号调控的功能机制仍不清楚。
体外原代神经元培养模型是一种简便、简便的模型, 可用于研究下丘脑神经元的胰岛素信号调节。在这项研究中, 我们介绍了 exogeneous GLUT4 蛋白与 GFP (GFP-GLUT4) 结合的主下丘脑神经元跟踪 GLUT4 膜易位后, 胰岛素刺激。GFP-GLUT4 蛋白贩运的时间推移图像的反褶积显微镜记录, 允许用户产生高速, 高分辨率的图像, 而不损害神经元显着进行实验。CCR5 在胰岛素调控的 GLUT4 易位中的作用在 CCR5 的下丘脑神经元中观察到, 从 CCR5 剔除小鼠中分离培养。结果表明, 胰岛素刺激后, CCR5 下丘脑神经元 GLUT4 膜移位效率降低。

免疫与下丘脑特异蛋白-亲 opiomelanocortin (POMC) 抗体和神经元标记微管相关蛋白 2 (MAP2) (图 2A) 进一步鉴别小鼠下丘脑神经元的培养。我们证实了主要培养下丘脑下丘脑蛋白 POMC 表达的神经元。CCR5 受体和 CCL5 在下丘脑神经元的表达与特异抗体和 POMC 抗体共同标记 (图 2A, 2B)。
经过3天的培养, 神经元转染 gfp DNA (图 3) 或 gfp 共轭 GLUT4 (图 4)。GFP 表达式通常可以在没有特定模式的单元格中找到 (图 3), 但 GLUT4-GFP 将在胞 (图 4) 中以类似于点状的结构表示。本研究采用的转染试剂盒不是最有效的神经元转染方法;然而, 这是一个较不严格的方法, 以更好的细胞存活后转染, 这有助于更好的活细胞成像/记录后。GFP-GLUT4 表达神经元的图像在时间推移电影 (图 4A-b, 补充视频 1, 2) 后, 在胰岛素刺激或 CCL5 刺激 (图 4C, 补充视频 3)。GFP 和 GFP-GLUT4 的信号在神经元中清晰而强烈。
下丘脑神经元与 GLUT4-GFP 转染进一步治疗胰岛素 (40 U) 的特点 GFP-GLUT4 贩运。Reprehensive 视频的 GLUT4-GFP 运动后, 胰岛素刺激在体重和 CCR5--下丘脑神经元分别显示为视频1和视频2。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56409/56409fig1.jpg"> 图 1: 在胚胎阶段 (16.5 天) 分离小鼠大脑不同区域的组织.(A-D)从胎盘分离幼崽所涉及的步骤。(E, F)从尸体上解剖一只幼崽的头部(G-I)从头骨隔离整个大脑所涉及的步骤。黑色的箭头指向要拉的方向, 而用镊子取出头骨。(J) 下丘脑的隔离。黑色箭头指向钳夹之间的下丘脑区域。(K L)鼠脑皮质的分离。黑色星号表示老鼠大脑的皮质区域, 白色箭头指向整个大脑皮质的分离。(M-o)海马部分与皮质的分离。上白色箭头标记海马组织, 下白色箭头标记皮层组织。刻度条 = 1 厘米 (A-F), 200 µm (G O)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/56409fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56409/56409fig2.jpg"> 图 2: 下丘脑神经元标记物 POMC 的特征和 CCL5 和 CCR5 的共同表达.(A) 原代培养的下丘脑神经元被标记为下丘脑神经元标记物-POMC (红色), CCR5 (绿色) 和神经元标记 MAP2 (灰色) 的共同表达。(B) POMC (红色) 下丘脑阳性神经元的 CCL5 (绿色) 表达 (根据参考文献17的补充数据改编)。在这里, DAPI 标记的细胞核与蓝色的颜色。在 (a) 和 (B) 中缩放条形图 = 50 µm. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/56409fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56409/56409fig3.jpg"> 图 3: GFP 蛋白在小鼠原代神经元中的表达.GFP 质粒 DNA 转染为4天培养后的原代培养神经元 (DIV4) 与脂质体, 并表示为另3天 (DIV7)。(A-b)GFP 在神经炎和躯体中均有表达。(C-D)模拟转染神经元;DAPI 将细胞核标记为 (B、D)。缩放栏 = 100 µm.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/56409fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56409/56409fig4.jpg"> 图 4: 下丘脑神经元 GFP-GLUT4 的快照.(A, C)GFP-GLUT4 蛋白表达于野生 (重量) 下丘脑神经元和 (B) CCR5/-下丘脑神经元。神经元被刺激与胰岛素 (A, B) 或 CCL5 (C)。箭头指向 GFP-GLUT4 点在神经炎之前 (-) 和之后 (+) 胰岛素或 CCL5 刺激和星号指向表面 GLUT4-GFP 之前 (-) 和后 (+) CCL5 刺激 (C).(图从引用17改编)。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/56409fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>5x Borade 缓冲器</td>
			<td>公司</td>
			<td>目录编号</td>
			<td>卷</td>
		</tr>
<tr>
<td>硼酸</td>
			<td>西格玛-爱秩序</td>
			<td>B6768</td>
			<td>1.55 克</td>
		</tr>
<tr>
<td>硼砂</td>
			<td>西格玛-爱秩序</td>
			<td>71997</td>
			<td>2.375 克</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td></td>
			<td></td>
			<td>100毫升</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td>过滤, 保持在4° c</td>
		</tr>
<tr>
<td>20x 聚 d-赖氨酸库存</td>
			<td>公司</td>
			<td>目录编号</td>
			<td>卷</td>
		</tr>
<tr>
<td>聚 d-赖氨酸</td>
			<td>西格玛-爱秩序</td>
			<td>P6407</td>
			<td>100毫克</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td></td>
			<td></td>
			<td>100毫升</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td>过滤, 保持在-20 ° c</td>
		</tr>
<tr>
<td>1x 聚 d-赖氨酸</td>
			<td></td>
			<td></td>
			<td>卷</td>
		</tr>
<tr>
<td>20x 聚 d-赖氨酸</td>
			<td></td>
			<td></td>
			<td>5毫升</td>
		</tr>
<tr>
<td>5x Borade 缓冲器</td>
			<td></td>
			<td></td>
			<td>20毫升</td>
		</tr>
<tr>
<td>ddH2O</td>
			<td></td>
			<td></td>
			<td>75毫升</td>
		</tr>
<tr>
<td>总</td>
			<td></td>
			<td></td>
			<td>100毫升</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td>保持在4° c</td>
		</tr>
<tr>
<td>洗涤介质</td>
			<td>公司</td>
			<td>目录编号</td>
			<td>卷</td>
		</tr>
<tr>
<td>DMEM-高葡萄糖</td>
			<td>gibco</td>
			<td>12800-017</td>
			<td>495毫升</td>
		</tr>
<tr>
<td>抗生素-Antimyotic</td>
			<td>gibco</td>
			<td>15240-062</td>
			<td>5毫升</td>
		</tr>
<tr>
<td>总</td>
			<td></td>
			<td></td>
			<td>500毫升</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td>保持在 4 C</td>
		</tr>
<tr>
<td>木瓜蛋白酶-胰蛋白酶消化缓冲液:</td>
			<td>公司</td>
			<td>目录编号</td>
			<td>容量或最后的集中</td>
		</tr>
<tr>
<td>木瓜蛋白酶 (10 毫克/毫升)</td>
			<td>西格玛-爱秩序</td>
			<td>P4762</td>
			<td>200µL (2 毫克/毫升)</td>
		</tr>
<tr>
<td>胰蛋白酶-EDTA (0.25%)</td>
			<td>gibco</td>
			<td>25200-072</td>
			<td>200µL (0.05%)</td>
		</tr>
<tr>
<td>洗涤介质</td>
			<td></td>
			<td></td>
			<td>600µL</td>
		</tr>
<tr>
<td>总</td>
			<td></td>
			<td></td>
			<td>1000µL</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td>保持在-20 ° c</td>
		</tr>
<tr>
<td>电镀介质:</td>
			<td>公司</td>
			<td>目录编号</td>
			<td>卷</td>
		</tr>
<tr>
<td>Neurobasal 培养基</td>
			<td>gibco</td>
			<td>21103-049</td>
			<td>176毫升</td>
		</tr>
<tr>
<td>胎牛血清</td>
			<td>gibco</td>
			<td>10437-028</td>
			<td>20毫升</td>
		</tr>
<tr>
<td>l-谷氨酸 (200 毫米)</td>
			<td>gibco</td>
			<td>25030</td>
			<td>2毫升</td>
		</tr>
<tr>
<td>抗生素-Antimyotic</td>
			<td>gibco</td>
			<td>15240-062</td>
			<td>2毫升</td>
		</tr>
<tr>
<td>总</td>
			<td></td>
			<td></td>
			<td>200毫升</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td>保持在4° c</td>
		</tr>
<tr>
<td>完整的培养基</td>
			<td>公司</td>
			<td>目录编号</td>
			<td>卷</td>
		</tr>
<tr>
<td>Neurobasal 培养基</td>
			<td>gibco</td>
			<td>21103-049</td>
			<td>95毫升</td>
		</tr>
<tr>
<td>N2 补充剂 (100x)</td>
			<td>gibco</td>
			<td>17502-048</td>
			<td>1毫升</td>
		</tr>
<tr>
<td>B27 补充剂 (50x)</td>
			<td>gibco</td>
			<td>17504-04</td>
			<td>2毫升</td>
		</tr>
<tr>
<td>l-谷氨酸 (200 毫米)</td>
			<td>gibco</td>
			<td>25030</td>
			<td>1毫升</td>
		</tr>
<tr>
<td>抗生素-Antimyotic</td>
			<td>gibco</td>
			<td>15240-062</td>
			<td>1毫升</td>
		</tr>
<tr>
<td>总</td>
			<td></td>
			<td></td>
			<td>100毫升</td>
		</tr>
<tr>
<td></td>
			<td></td>
			<td></td>
			<td>新鲜的准备</td>
		</tr>
</tbody></table>表 1: 本研究中使用的消化缓冲液和培养基成分。
补充视频 1:胰岛素刺激下丘脑神经元 GFP-GLUT4 运动.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/JoVE-Video-1.mov">请单击此处下载此文件.</a>
补充视频 2:胰岛素刺激 GFP-GLUT4 运动在 CCR5-/-下丘脑神经元.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/JoVE-Video-2.mov">请单击此处下载此文件.</a>
补充视频 3:CCL5 刺激下丘脑神经元的 GFP-GLUT4 运动(视频从引用17改编)。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56409/JoVE-Video-3.mov">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy at JoVE.com

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

本协议描述了一种实时绿色荧光蛋白 (GFP) 标记葡萄糖转运蛋白 4 (GLUT4) 蛋白质贩运的技术, 对胰岛素刺激和 CCR5 在 insulin–GLUT4 的生物学作用的表征带反褶积显微镜的信号通路。

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

live-images-glut4-protein-trafficking-mouse-primary-hypothalamic

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.6K 次观看.  Taipei Medical University. 本协议描述了一种实时绿色荧光蛋白 (GFP) 标记葡萄糖转运蛋白 4 (GLUT4) 蛋白质贩运的技术, 对胰岛素刺激和 CCR5 在 insulin–GLUT4 的生物学作用的表征带反褶积显微镜的信号通路。

视频: Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

在原代培养海马神经元的密集核心囊泡的实时成像

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

科研

生物学

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

神经科学

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

流式细胞仪定量测定GLUT4转位到质膜

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx due to the abnormal activation of glutamate receptors results in DA excitotoxicity and has been identified as an important mechanism for DA neuron loss. In this study, we isolate, dissociate, and culture midbrain neurons from the mouse ventral mesencephalon (VM) of ED14 mouse embryos. We then infect the long-term primary mouse midbrain cultures with an adeno-associated virus (AAV) expressing a genetically encoded calcium indicator, GCaMP6f under control of the human neuron-specific synapsin promoter, hSyn. Using live confocal imaging, we show that cultured mouse midbrain neurons display spontaneous Ca2+ fluxes detected by AAV-hSyn-GCaMP6f. Bath application of glutamate to midbrain cultures causes abnormal elevations in intracellular Ca2+ within neurons and this is accompanied by caspase-3 activation in DA neurons, as demonstrated by immunostaining. The techniques to identify glutamate-mediated apoptosis in primary mouse DA neurons have important applications for the high content screening of drugs that preserve DA neuron health.

Quantifying Spontaneous Ca2+ Fluxes and their Downstream Effects in Primary Mouse Midbrain Neurons

Here we present a protocol to measure in vitro Ca2+ fluxes in midbrain neurons and their downstream effects on caspase-3 using primary mouse midbrain cultures. This model can be employed to study pathophysiologic changes related to abnormal Ca2+ activity in midbrain neurons, and to screen novel therapeutics for anti-apoptotic properties.

定量自发 Ca2+ 通量及其在原发性小鼠中脑神经元中的下游效应

Parkinson&#8217;s disease (PD) is a devastating neurodegenerative disorder caused by the degeneration of dopaminergic (DA) neurons. Excessive Ca2+ influx ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical manifestations include weight loss, dyslipidemia, and hypermetabolism; however, it remains unclear how these relate to motor neuron degeneration. Using a Drosophila model of TDP-43 proteinopathy that recapitulates several features of ALS including cytoplasmic inclusions, locomotor dysfunction, and reduced lifespan, we recently identified broad ranging metabolic deficits. Among these, glycolysis was found to be upregulated and genetic interaction experiments provided evidence for a compensatory neuroprotective mechanism. Indeed, despite upregulation of phosphofructokinase, the rate limiting enzyme in glycolysis, an increase in glycolysis using dietary and genetic manipulations was shown to mitigate locomotor dysfunction and increased lifespan in fly models of TDP-43 proteinopathy. To further investigate the effect on TDP-43 proteinopathy on glycolytic flux in motor neurons, a previously reported genetically encoded, FRET-based sensor, FLII12Pglu-700&#181;&#948;6, was used. This sensor is comprised of a bacterial glucose-sensing domain and cyan and yellow fluorescent proteins as the FRET pair. Upon glucose binding, the sensor undergoes a conformational change allowing FRET to occur. Using FLII12Pglu-700&#181;&#948;6, glucose uptake was found to be significantly increased in motor neurons expressing TDP-43G298S, an ALS causing variant. Here, we show how to measure glucose uptake, ex vivo, in larval ventral nerve cord preparations expressing the glucose sensor FLII12Pglu-700&#181;&#948;6 in the context of TDP-43 proteinopathy. This approach can be used to measure glucose uptake and assess glycolytic flux in different cell types or in the context of various mutations causing ALS and related neurodegenerative disorders.

Measuring Glucose Uptake in Drosophila Models of TDP-43 Proteinopathy

Glucose uptake is increased in Drosophila motor neurons affected by TAR DNA binding protein (TDP-43) proteinopathy, as indicated by a FRET-based, genetically encoded glucose sensor.

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

Amyotrophic lateral sclerosis is a neurodegenerative disorder causing progressive muscle weakness and death within 2-5 years following diagnosis. Clinical ...

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

This article describes a protocol to determine differences in basal redox state and redox responses to acute perturbations in primary hippocampal and cortical neurons using confocal live microscopy. The protocol can be applied to other cell types and microscopes with minimal modifications.

使用比率指标对原代神经元中的线粒体谷胱甘肽氧化还原状态进行实时成像

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant ...

<meta charset="utf8"></head><body>通过活细胞成像可视化原代海马神经元中的蛋白质

Using Live&#45;Cell Imaging to Measure the Effects of Pathological Proteins on Axonal Transport in Primary Hippocampal Neurons

Bidirectional transport of cargos along the axon is critical for maintaining functional synapses, neural connectivity, and healthy neurons. Axonal transport is disrupted in multiple neurodegenerative diseases, and projection neurons are particularly vulnerable because of the need to transport cellular materials over long distances and sustain substantial axonal mass. Pathological modifications of several disease-related proteins negatively affect transport, including tau, amyloid-&#946;, &#945;-synuclein, superoxide dismutase, and huntingtin, providing a potential common mechanism by which pathological proteins exert toxicity in disease. Methods to study these toxic mechanisms are necessary to understand neurodegenerative disorders and identify potential therapeutic interventions.
Here, cultured primary rodent hippocampal neurons are co-transfected with multiple plasmids to study the effects of pathological proteins on fast axonal transport using live-cell confocal imaging of fluorescently-tagged cargo proteins. We begin with the harvest, dissociation, and culturing of primary hippocampal neurons from rodents. Then, we co-transfect the neurons with plasmid DNA constructs to express fluorescent-tagged cargo protein and wild-type or mutant tau (used as an exemplar of pathological proteins). Axons are identified in live cells using an antibody that binds an extracellular&#160;domain of neurofascin, an axon initial segment protein, and an axonal region of interest is imaged to measure fluorescent cargo transport.
Using KymoAnalyzer, a freely available ImageJ macro, we extensively characterize the velocity, pause frequency, and directional cargo density of axonal transport, all of which may be affected by the presence of pathological proteins. Through this method, we identify a phenotype of increased cargo pause frequency associated with the expression of pathological tau protein. Additionally, gene-silencing shRNA constructs can be added to the transfection mix to test the role of other proteins in mediating transport disruption. This protocol is easily adaptable for use with other neurodegenerative disease-related proteins and is a reproducible method to study the mechanisms of how those proteins disrupt axonal transport.

<meta charset="utf8"></head><body>作者聚焦：了解病理蛋白对神经退行性疾病轴突转运的影响

Here, we demonstrate how to combine transfection of primary hippocampal rodent neurons with live-cell confocal imaging to analyze pathological protein-induced effects on axonal transport and identify mechanistic pathways mediating these effects.

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

摘要

探索更多视频

此视频中的章节

在原代培养海马神经元的密集核心囊泡的实时成像

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

流式细胞仪定量测定GLUT4转位到质膜

定量自发 Ca²⁺ 通量及其在原发性小鼠中脑神经元中的下游效应

小鼠视网膜的经瞳双光子体内成像

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取

使用比率指标对原代神经元中的线粒体谷胱甘肽氧化还原状态进行实时成像

使用活细胞成像测量病理蛋白对原代海马神经元轴突运输的影响

使用活细胞成像测量病理蛋白对原代海马神经元轴突运输的影响

使用活细胞成像测量病理蛋白对原代海马神经元轴突运输的影响

用反褶积显微镜观察小鼠原发性下丘脑神经元 GLUT4 蛋白的动态图像

摘要

探索更多视频

此视频中的章节

在原代培养海马神经元的密集核心囊泡的实时成像

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

流式细胞仪定量测定GLUT4转位到质膜

定量自发 Ca2+ 通量及其在原发性小鼠中脑神经元中的下游效应

小鼠视网膜的经瞳双光子体内成像

测量TDP-43蛋白病果 蝇 模型中的葡萄糖摄取

使用比率指标对原代神经元中的线粒体谷胱甘肽氧化还原状态进行实时成像

使用活细胞成像测量病理蛋白对原代海马神经元轴突运输的影响

使用活细胞成像测量病理蛋白对原代海马神经元轴突运输的影响

使用活细胞成像测量病理蛋白对原代海马神经元轴突运输的影响

定量自发 Ca²⁺ 通量及其在原发性小鼠中脑神经元中的下游效应

测量TDP-43蛋白病果蝇模型中的葡萄糖摄取