Name: in vivo single-molecule tracking at the drosophila presynaptic motor nerve terminal
Uploaded: 2018-01-14T14:00:00.000+00:00
Duration: 6 min 45 s
Description: Watch this Scientific Journal Video about In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal at JoVE.com

我们感谢吉恩-巴蒂斯特 Sibarita 和丹尼尔 Choquet (研究所, CNRS/波尔多大学) 对分子分析的支持。我们特别感谢尼克 Valmas 和杰西卡麦高 (QBI, 昆士兰大学) 为他们的帮助与视频录制, 配音以及图形的平面设计。这项工作得到澳大利亚研究理事会 (AR) 发现项目 (DP170100125 至 F.A.M.)、澳大利亚研究理事会 (欣喜赠款 LE0882864 至 F.A.M.)、未来研究金 (FT100100725 至 B.V.S.)、澳大利亚研究理事会 (欣喜赠款LE130100078 至 F.A.M.)和澳洲项目补助金 (APP1103923 到 B.V.S.)。F.A.M. 是一位澳洲的高级研究员 (ONT1060075)。

1. 转基因果蝇表达 mEos2-Tagged 蛋白的设计
<ol><li>选择要用 mEos2 蛋白标记的蛋白质。为了提高成像成功率, 在神经元等离子膜中选择具有跨膜域的蛋白质14 (例如、Syntaxin-1A)、突触泡上的蛋白质或体26、27 (如Synaptobrevin-2), 或胞浆蛋白与神经元细胞膜蛋白 (例如, Munc18-1) 密切互动。</li>
	<li>构造转基因编码 mEos2-tagged 蛋白 (图 1)。设计了蛋白质和 mEos2 的正反引物。 注意: mEos2 编码序列可以从 pmEos2-N1 质粒中放大, 这是为了融合到感兴趣的蛋白质的 c-末端。例如, 克隆可以通过在体内重组在酿酒酵母中使用 pFL44S-attB-MCS-w+ 向量14,28, 交替使用其他克隆方法, 如吉布森程序集协议29。<ol>
<li>线性将 BamHI 和 XbaI 和 co-transform 的向量与ura3-酵母细胞结合, 并结合 PCR 片段编码 mEos2 和感兴趣的蛋白质。注入果蝇胚胎与构造生成一个转基因飞线30。</li>
	</ol>
</li>
	<li>在标准酵母和糖蜜培养基上或在塑料瓶中含有的其他合适的替代品后, 将转基因果蝇培养起来。为了确保健康和相当大的突触 boutons31, 避免在小瓶中过度拥挤, 并在每天产卵后将苍蝇翻转到新的瓶子里;这可确保第三龄幼虫在开始在培养基上游荡时, 能很好地喂养并达到适当的大小。在室温下提高转基因蝇 (25 ° c)。 注: 较大的突触 boutons 倾向于在成像过程中产生更大数量的可视化蛋白质。</li>
</ol>2. 第三龄幼虫解剖
<ol><li>使用合适的硅胶弹性体套件 (图 2A), 使直径为≤20毫米的圆柱形或半圆形型烷 (聚硅氧烷) 底座和≤20 mm 的高度。混合硅酮弹性体与硅硬化剂的比例为10:1。<ol>
<li>将混合物彻底搅拌5分钟, 将混合物倒入模具中, 上面列出的 dimsensions。让它在烤箱硬化在100° c 为45分钟。该基地将作为进行解剖的基础。确保与玻璃底培养皿的井相配合。</li>
	</ol>
</li>
	<li>从小瓶壁上选择一个游荡的第三龄幼虫。使用光学立体显微镜在4.5 倍放大, 以可视化的幼虫放置在与背面的硅橡胶圆柱形基地。</li>
	<li>使用弯曲和角度的镊子, 坚持 minutien 针头上的嘴钩, 两个延长前气孔之间, 并在尾部的肛门, 两个后气孔。在室温下将施耐德的昆虫培养基加40µL 到固定的幼虫上。 注意: Hemolyph 类似的解决方案 (HL3 或 HL6) 也可以在施耐德的解决方案3233的位置使用。</li>
	<li>通过使用 minutien 针将幼虫腹壁的背侧切开进入壁的中线。</li>
	<li>使用弹簧剪刀从切口点切开身体壁。沿中线在前面后方方向切开34。</li>
	<li>用细钳和弹簧剪刀破幼虫: 去除内脏, 包括唾液腺、气管和内脏。用施耐德的昆虫培养基冲洗两次幼虫。</li>
	<li>用细钳, 按住两个身体壁襟翼, 轻轻地伸展出来, 并坚持两个 minutien 针两侧。这将产生一个六边形形状的准备 (图 2A)。</li>
	<li>用弹簧剪刀将大脑从腹侧神经线上分离出来, 以减少成像过程中肌肉运动的可能性;这也有助于保持准备单位对成像盘。使用弯曲的镊子将所有六 minutien 引脚推入到该基地。 注: 只有一小部分的引脚应嵌入在腹壁内。</li>
	<li>为了证实幼虫在解剖过程中幸存下来, 轻微地戳腹神经线, 这应该引出幼虫腹壁的蠕动收缩。</li>
</ol>3. 微斜光照单粒子跟踪显微镜
<ol><li>将被解剖的幼虫--硅橡胶底座倒置到玻璃底的培养皿中。用2毫升室温施耐德的昆虫培养基填充成像盘 (图 2B)。</li>
	<li>使用 63 x/1.2 NA 浸水目标在荧光全内反射荧光 (TIRF) 显微镜下, 在腹部肌肉6和 735中找到第二和第三段的运动神经元。</li>
	<li>使用显微镜的目镜定位解剖幼虫;使用明亮的磁场照明识别肌肉6。</li>
	<li>使用显微镜采集软件 (参见材料表), TIRF 配置;将准直摄像机的角度滑动至°, 增加穿透深度, 否则 TIRF 临界角移出以达到斜向照明。</li>
	<li>开关 488 nm 激光, 以可视化 mEos2 在绿色。使用低激光功率 (少于5% 的22.8 兆瓦), 以避免漂白绿色荧光。找到神经肌肉结 (看起来像一个字符串上的珠子), 并获得低分辨率 TIRF 图像。</li>
	<li>同时开关 405 nm 激光 (photoconverts mEos2 从绿色到红色物种) 和 561 nm 激光 (图像 photoconverted 红色 mEos2) 随机定位单一 mEos2 荧光蛋白。 注: 建议使用低 405 nm 激光功率 (0.005 到0.9% 的4.78 兆瓦), 因为这允许在电影采集期间相对恒定的 photoconversion 率。使用50和75% 的 561 nm 激光 (20.1 兆瓦), 因为这是足以获得荧光从红 mEos2 物种, 而不影响肌肉组织的形态学周围的神经肌肉结。</li>
	<li>对于每个神经肌肉结链, 获得一个时间序列由1.5万帧或更多的33赫兹. 保存为. czi 格式, 以保留所附的元数据以供可能参考。</li>
	<li>使用 ImageJ 将 ". czi" 电影文件转换为 ". tiff" 文件。</li>
	<li>用合适的分子跟踪分析软件分析获得的电影11,14,36 , 如 TrackMate 在斐济/ImageJ37 (参见材料表)。通过灰度点扩展函数的高斯拟合, 定位每个定位的 "x" 和 "y" 坐标。 注:疑难解答: 如果在接触 488 nm 激光时未观察到绿色荧光或在神经肌肉接合处太暗, 则简要切换 photoconverting 和成像激光器 (405 nm 和 561 nm), 因为这有助于产生更多的红色mEos2 的种类。然后切换回 488 nm 激光器, 并采取 TIRF 图像。</li>
</ol>4. 固定幼虫的单分子定位
<ol><li>通过手掌来测量蛋白质簇的大小, 在解剖后用固定剂-4% 甲醛稀释磷酸盐缓冲盐 (PBS) 取代施耐德的昆虫培养基。</li>
	<li>在室温下孵育幼虫30分钟。</li>
	<li>取出解剖针脚, 并将固定的幼虫放在1.7 毫升的透明锁离心管中。</li>
	<li>根据需要解剖和修复其他许多幼虫, 然后用 PBS 冲洗三次。</li>
	<li>将 pbs 替换为块缓冲 (pbs、0.1% 卫 X-100 和3% 正常山羊血清的溶液)。</li>
	<li>用 PBS 三次冲洗幼虫, 然后用必要的主抗体 (在块缓冲液中稀释) 进行孵育。</li>
	<li>在 4 oC 夜间孵育。</li>
	<li>用 PBS 洗涤幼虫三次, 然后用必要的二级抗体孵育 (稀释在块缓冲液中)。</li>
	<li>用 PBS 清洗幼虫三次, 将幼虫附着在其上, 并像先前描述的分子跟踪一样。</li>
</ol>

作者没有什么可透露的。

该协议描述了在果蝇第三龄幼虫的神经肌肉交界处的 mEos2-tagged 蛋白的分子跟踪。为了避免转基因蛋白的表达, Syntaxin-1A-mEos2 表达是由内源性 Syntaxin-1A 启动子驱动的。突触蛋白迁移的研究主要限于体外培养细胞和皮层神经元的调查9,11,12,13。这些蛋白质是否在活体动物中表现出类似的移动特征, 这在很大程度上是未知的。要可视化 single-protein 的移动性在体内, 必须克服诸如光学透明度、可访问性和穿透深度等限制。通过解剖幼虫的准备和可视化肌肉表面的神经肌肉连接, 我们避免了光学透明度和可达性问题。试图在正常的 TIRF 配置中映像分子移动的尝试被证明是不成功的。然而, 使用轻微倾斜照明允许增加激发激光穿透深度。此外, 用于固定幼虫的 minutien 针有助于避免麻醉使用对蛋白质流动性可能产生的不利影响。可以使用更高的放大目标, 如100x 油浸的目标, 以可视化单个分子在体内。但是, 放大倍数的增加可能会增加漂移的发生。此外, 我们使用了较低的强度 (〜 30%)561 nm 激光成功地在运动神经终端图像单分子。使用较低的激光功率可能需要额外的测试。
本协议适用于可视化的突触蛋白在神经元细胞膜附近的移动性。使用这种方法, 诱捕蛋白-Syntaxin-1A 和 autophagosome 绑定蛋白 Atg18a 的移动性已经成功地成像在体内14,26。在运动神经元轴突上的蛋白质的流动性也可以被调查27。然而, 由于底层组织产生的高背景信号, 对大脑或腹神经线上蛋白质的移动性的研究可能难以评估;此外, 可视化蛋白质的流动性在同一大脑结构将需要荧光标记的大脑结构 (以确保可), 并使用双摄像头成像将是必要的。
目前的方法可用于超分辨率显微镜在果蝇主要限于成像胚胎, 而不是更发达的第三龄幼虫38,39。这些协议的主要问题是它们在图像区域中产生的轨迹数很低, 因此防止了移动状态的 in-depth 分析22,40。我们的在体内单分子跟踪协议有能力产生大量的弹道, 因此可以用于其他动物模型, 如Caenorhabiditis 线虫和斑马鱼。事实上, 每个 NMJ 链生成了2400个轨迹, 使得它适合于分析这些状态1427之间不同的移动状态和转换。此外, 许多体内单分子成像策略依赖于使用麻醉剂来固定动物20,22, 这可能会改变成像完成的生理功能。在这里, 我们优化了 ' 无麻醉 ' 协议, 以固定的幼虫使用 minutien 针。
这项协议的潜在用途可能是在三维度中可视化蛋白质的移动性。超分辨率显微镜的最新进展使蛋白质的移动性可以在 "x"、"y" 和 "z" 维度的体外可视化,41,42。我们目前的方法并没有考虑到 "z" 轴中蛋白质的流动性。然而,果蝇运动神经终端是一个球形结构, 未来有价值的方法将是在三维卷43中量化蛋白质的移动性。在 z 维的成像是可行的使用散光透镜。或者, 在 TIRF 模式44、45中, z 分辨率也会增加。然而, 在我们的实验中, 我们使用了略微倾斜的光照来可视化单分子的 syntaxin1A-mEos2, 而不需要散光透镜。
总之, 这两种转基因果蝇的可用性都是以 photoconvertible 蛋白为标志的蛋白质, 它是一个用于解剖转基因幼虫的 TIRF 显微镜, 同时也有可能改变光照角度是本协议中所描述的单个蛋白质的可视化所需的最重要的工具。

神经元通信依赖神经递质释放, 这是通过调节融合的神经递质包含突触泡与等离子膜1。此过程称为胞2、3、4、5、6是高度动态的, 可以根据刺激速率的波动进行上调或下调7。这些过程中所涉及的蛋白质受布朗运动的制约, 因此显示纳米组织能够维持一系列的结合行动, 支撑这些生理功能。细胞膜蛋白是高度动态的, 允许分子在等离子膜上簇的侧向俘获7,8,9,10,11, 12。因此, 调查这些蛋白质的流动性有助于阐明它们的操作模式8。突触蛋白, 如可溶性的 n-ethylmaleimide 敏感因子附着蛋白受体 (圈套), 例如 Syntaxin-1A, 现在已知的横向快速移动, 以及横向诱捕簇内可能作为分子用于囊泡融合的仓库和站点9,13,14,15。
最近发展的 photoconvertible 荧光蛋白, 如单体 (m) Eos16,17和枫18, 通过使用这种方法, 使纳米的神经元细胞膜蛋白的分辨率作为相片激活的定位显微镜 (PALM) 和随机光学重建显微镜 (风暴)14,15,19。这些发展使得对生物蛋白在培养的活细胞和神经元中的流动性和 nanoclustering 性进行了调查。最近, 已进行了研究, 以阐明 (在类似的高分辨率) 这些膜蛋白的动力学和组织的活完整的生物体神经元, 如小家鼠,线虫线虫,和果蝇14,20,21,22。
调查蛋白质的动力学和组织在体内不仅需要使用最近开发的超分辨率成像工具 (如手掌, 风暴, photoconvertible 荧光), 而且还有能力克服障碍, 如作为成像激光的蛋白质和穿透深度的可及性。图像中的细胞内蛋白在一个完整的动物是固有的挑战, 因为是成像蛋白质的结构深埋在活体组织的完整的动物, 如海马的完整的鼠标。因此, 在或接近组织表面的蛋白质更容易成像。在体内成像的另一个困难是确保在动物之间重现性。由于解剖可能不同, 从一个样本到另一个, 选择一个结构与易于复制在不同的完整动物标本也可能证明具有挑战性。使用诸如突触之类的定型结构有助于克服这些困难。
最近的研究已经在动物身上成像了蛋白质的流动性, 例如线虫线虫22等光学透明的表皮, 而其他的调查则在组织/器官的表面上成像蛋白质组织肌动蛋白成像的皮层神经元通过头骨的活鼠21。在某些情况下, 麻醉剂被用来保持动物不动;然而, 特定的麻醉剂可能会对感兴趣的蛋白质产生不利影响。因此, 应探索在活体中保持动物静止的替代方法, 以尽量减少错误。
对超分辨率成像的另一个警告是在体内是用来照亮感兴趣的蛋白质的激光器会对周围的组织产生不利的影响, 因此建议尽量保持尽可能低的激发强度。激光照射周围组织的光照也会增加背景信号。低激发强度的使用有助于降低背景, 警告是它也可能导致荧光蛋白光子的产量下降。在分析过程中, 背景减法可以作为一种替代方法, 在图像分析之前减少背景信号。
果蝇为活体成像提供了一种理想的生物体, 因为它为研究特定的蛋白质功能的遗传学工具奠定了良好的基因。上游激活序列 (无人 Gal4 系统), 使蛋白质的时间和空间表达, 因此可以用来操纵神经23, 这样, 热遗传刺激使用果蝇瞬态受体电位 sub-family A1 (dTRPA1)24或使用远红移 CsChrimson25的光遗传刺激可以与成像14组合。我们克服了在这个系统中执行体内分子成像的许多并发症, 现在描述如何在果蝇中进行单跟踪。

<table><tbody><tr><td>PDMS (Sylgard 184 silicone elastomer kit)</td><td>Dow Corning Corporation</td><td>000000000001064291</td><td></td></tr><tr><td>Glass-bottomed Culture Dish</td><td>In Vitro Scientific</td><td>D29-20-1.5-N</td><td></td></tr><tr><td>Optical Stereo-microscope</td><td>Olympus</td><td>SZ51</td><td></td></tr><tr><td>Curved Tweezers (Style 5/45)</td><td>Electron Microscopy Sciences</td><td>0208-5AC-PO</td><td></td></tr><tr><td>Minutien Pins (0.1mm)</td><td>Fine Science Tools</td><td>26002-10</td><td></td></tr><tr><td>Schneider&#039;s Insect Medium</td><td>Sigma, Life Sciences</td><td>S0146</td><td></td></tr><tr><td>Vannas Spring Scissors</td><td>Fine Science Tools</td><td>15000-00</td><td></td></tr><tr><td>Fine Forceps (Dumont #5)</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>ELYRA PS.1 Microscope</td><td>Zeiss</td><td>PS.1</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Lonza</td><td>17-515Q</td><td></td></tr><tr><td>Paraformaldehyde (16%)</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T-9284</td><td></td></tr><tr><td>Normal Goat Serum</td><td>Sigma-Aldrich</td><td>G9023</td><td></td></tr><tr><td>Maxyclear snaplock microcentrifuge Tube (1.7 mL)</td><td>Axygen</td><td>MCT-175-C</td><td></td></tr><tr><td>Zen Black acquisition software</td><td>Carl Zeiss, 2012</td><td></td><td></td></tr><tr><td>Metamorph software</td><td>Molecular Devices</td><td>7.7.8</td><td>PALM Tracer Plugin</td></tr><tr><td>Fly strain</td><td></td><td>w&lt;sup&gt;1118&lt;/sup&gt;; HA-Sx1A-mEos2</td><td></td></tr></tbody></table>

in vivo single-molecule tracking at the drosophila presynaptic motor nerve terminal

越来越多的超分辨率显微镜技术正在帮助揭开控制纳米细胞世界的机制。单分子成像是获得动力, 因为它提供了独特的机会, 可视化的单个分子在活细胞。在这里, 我们描述了一种技术, 我们开发了执行单跟踪照片激活的定位显微镜 (sptPALM) 在果蝇幼虫。突触通信依赖于关键的前蛋白, 通过对接、启动和促进与细胞膜的神经递质囊泡的融合。一系列蛋白质蛋白质和蛋白质-脂质相互作用严密调控这些过程和前蛋白质因此表现出变动与每一个这些关键事件相关。研究这些蛋白质的流动性如何与它们在完整的活体动物中的生理功能相互关联, 对于了解它们的确切作用机制至关重要。提取蛋白质的流动性高分辨率在体内需要克服的限制, 如光学透明度, 可访问性和穿透深度。我们描述如何 photoconvertible 荧光蛋白标记的前蛋白 Syntaxin-1A 可以可视化通过轻微的倾斜照明和跟踪在电机神经终端或沿运动神经轴突的第三龄果蝇幼虫.

使用上面所列的协议, 生成了 Syntaxin-1A-mEos2 的转基因Drosophilamelanogaster (图 1)。转基因第三龄果蝇幼虫在硅橡胶圆柱形底座上解剖 (图 2AF)。为了直观地显示单分子的 Syntaxin-1A, 我们在 TIRF 显微镜上用手掌稍微倾斜激光照明14。
在第二腹段肌肉6的前运动神经末端上追踪 Syntaxin-1A 的单分子。在使用 488 nm 激光 (图 3A) 时, mEos2 的低分辨率图像可以检测出 Syntaxin-1A-mEos2 在 1b boutons 上的绿色荧光。在成像实验中, 405 nm 和 561 nm 的激发激光成像深度分别为 133.5 nm 和 165.6 nm。绿色图像作为平均16个人帧获得。这个绿色的图像被用来创造感兴趣的区域, 用来限制分析定位在 photoconverted 电影的前运动神经终端单独。
对获得的 photoconverted sptPALM 电影的分析产生了构强度、扩散系数和轨迹图 (图 3A)。通过分析单个轨迹的均方位移 (图 3B), 进一步量化了 Syntaxin-1A-mEos2 的移动性。统计比较可以使用在曲线 (图 3B) 下的区域进行。移动性的进一步分析产生了 Syntaxin-1A-mEos2 的扩散系数分布, 这可以通过将流动与不动种群 (图 3C) 的比较来进行统计评估。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56952/56952fig1.jpg"> 图 1: 生成 mEos2-tagged 构造.pFL44S-attB-MCS-w+ 向量是用 BamHI 和 XbaI 线性化的。重叠聚合酶链反应 (PCR) 片段编码的兴趣蛋白 (蒲) 和 mEos2, 分别, 可以克隆, 例如, 通过在体内重组在ura3-酵母细胞或吉布森组装。请注意, 为了产生 Syntaxin-1A-mEos2 的转基因, 我们克隆的结构两侧的内生 Syntaxin-1A 启动器和终止序列。由此产生的结构可以随后被注入到果蝇胚胎中, 以创建一条表达感兴趣的蛋白 (mEos2) 的转基因飞线。<a href="//cloudflare.jove.com/files/ftp_upload/56952/56952fig1large.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/56952/56952fig2.jpg"> 图 2:果蝇 幼虫解剖和倾斜的照明。(A) 模式显示果蝇幼虫解剖在 half-cylindrical 的基础上进行。通过使用 minutien 针, 鱼片幼虫被保存在了硅橡胶凝胶上。(B-C)幼虫--硅橡胶的准备是放置在一个玻璃底培养皿中含有施耐德的昆虫培养基。TIRF 显微镜在轻微倾斜的照明配置是用来可视化 Syntaxin-1A-mEos2 在电机神经终端。(d-F)一个 half-cylindrical 的硅橡胶基地, 解剖了果蝇幼虫。幼虫--硅橡胶的制备被放置在一个充满施耐德培养基的成像盘中, 幼虫在超分辨手掌显微镜上被影像。刻度栏, 10 mm (此图已从14中修改)。<a href="//cloudflare.jove.com/files/ftp_upload/56952/56952fig2large.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/56952/56952fig3.jpg"> 图 3:在体内跟踪 Syntaxin-1A-mEos2 在前马达神经末端。(a) 在1b 型运动神经终端上的低分辨率 TIRF 图像 Syntaxin-1A-mEos2。分析的 photoconversion 电影成像在 33 Hz 产生的 sptPALM 构强度, 扩散系数, 弹道图。5309个单独的轨迹被成像超过1.6万帧电影采集。刻度条, 3 μ m。(B-C)从6个不同的运动神经终端绘制了 Syntaxin-1A-mEos2 的均方位移图;在 "曲线" (联合自卫队) 下的区域被用来量化移动的水平。扩散系数分布揭示了流动和静止的 Syntaxin-1A 种群, 可以量化为彼此的比值;每个运动神经末端 (n = 3 幼虫) 平均获得2400弹道。数据显示为平均值的标准误差。<a href="//cloudflare.jove.com/files/ftp_upload/56952/56952fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal at JoVE.com

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

在体内果蝇前运动神经末端的单分子跟踪

在这里, 我们说明如何单分子照片激活定位显微镜可以进行的运动神经终端的一个活的果蝇第三龄幼虫。

An increasing number of super-resolution microscopy techniques are helping to uncover the mechanisms that govern the nanoscale cellular world. ...

in-vivo-single-molecule-tracking-at-drosophila-presynaptic-motor

Research

JoVE Journal

Neuroscience

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

8.4K 次观看.  The University of Queensland. 在这里, 我们说明如何单分子照片激活定位显微镜可以进行的运动神经终端的一个活的果蝇第三龄幼虫。

视频: In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different neurological diseases. Defects in this essential process can have detrimental effects on neuronal functioning and development. We have developed a dissection protocol that is designed to expose the Drosophila larval segmental nerves to view axonal transport in real time. We have adapted this protocol for live imaging from the one published by Hurd and Saxton (1996) used for immunolocalizatin of larval segmental nerves. Careful dissection and proper buffer conditions are critical for maximizing the lifespan of the dissected larvae. When properly done, dissected larvae have shown robust vesicle transport for 2-3 hours under physiological conditions. We use the UAS-GAL4 method 1 to express GFP-tagged APP or synaptotagmin vesicles within a single axon or many axons in larval segmental nerves by using different neuronal GAL4 drivers. Other fluorescently tagged markers, for example mitochrondria (MitoTracker) or lysosomes (LysoTracker), can be also applied to the larvae before viewing. GFP-vesicle movement and particle movement can be viewed simultaneously using separate wavelengths.

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

This protocol discusses the live dissection of Drosophila larvae for the purpose of imaging the movement of GFP tagged axonal vesicles on microtubule tracks.

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

科研

神经科学

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

发育生物学

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

在完整的果蝇幼虫体内的亚细胞分辨率成像

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

生物学

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

一个简单的神经元机械损伤方法学研究<em&gt;果蝇</em&gt;运动神经元变性

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding environment and initiate contacts with potential synaptic partners. Although the connection between dendritic branch dynamics and synaptogenesis is well established, how developmental and activity-dependent processes regulate dendritic branch dynamics is not well understood. This is partly due to the technical difficulties associated with the live imaging and quantitative analyses of these fine structures using an in vivo system. We established a method to study dendrite dynamics using Drosophila larval ventral lateral neurons (LNvs), which can be individually labeled using genetic approaches and are accessible for live imaging. Taking advantage of this system, we developed protocols to capture branch dynamics of the whole dendritic arbor of a single labeled LNv through time-lapse live imaging. We then performed post-processing to improve image quality through drift correction and deconvolution, followed by analyzing branch dynamics at the single-branch level by annotating spatial positions of all branch terminals. Lastly, we developed R scripts (Supplementary File) and specific parameters to quantify branch dynamics using the coordinate information generated by the terminal tracing. Collectively, this protocol allows us to achieve a detailed quantitative description of branch dynamics of the neuronal dendritic arbor with high temporal and spatial resolution. The methods we developed are generally applicable to sparsely labeled neurons in both in vitro and in vivo conditions.

Time-lapse Live Imaging and Quantification of Fast Dendritic Branch Dynamics in Developing Drosophila Neurons

Here, we describe the method we employed to image highly motile dendritic filopodia in a live preparation of the Drosophila larval brain, and the protocol we developed to quantify time-lapse 3D imaging datasets for quantitative assessments of dendrite dynamics in developing neurons.

发展果蝇神经元的快速丹德林分体动力学的延时实时成像和定量

Highly motile dendritic filopodia are widely present in neurons at early developmental stages. These exploratory dynamic branches sample the surrounding ...

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. Furthermore, despite progress in understanding postsynaptic MTs, much less is known about the contributions of presynaptic MTs to neuronal morphogenesis. In particular, studies of in vivo MT dynamics in Drosophila sensory dendrites yielded significant insights into polymer-level behavior. However, the technical and analytical challenges associated with live imaging of the fly neuromuscular junction (NMJ) have limited comparable studies of presynaptic MT dynamics. Moreover, while there are many highly effective software strategies for automated analysis of MT dynamics in vitro and ex vivo, in vivo data often necessitate significant operator input or entirely manual analysis due to inherently inferior signal-to-noise ratio in images and complex cellular morphology. &#160;To address this, this study optimized a new software platform for automated and unbiased in vivo particle detection. Multiparametric analysis of live time-lapse confocal images of EB1-GFP labeled MTs was performed in both dendrites and the NMJ of Drosophila larvae and found striking differences in MT behaviors. MT dynamics were furthermore analyzed following knockdown of the MT-associated protein (MAP) dTACC, a key regulator of Drosophila synapse development, and identified statistically significant changes in MT dynamics compared to wild type. These results demonstrate that this novel strategy for the automated multiparametric analysis of both pre- and postsynaptic MT dynamics at the polymer-level significantly reduces human-in-the-loop criteria. The study furthermore shows the utility of this method in detecting distinct MT behaviors upon dTACC-knockdown, indicating a possible future application for functional screens of factors that regulate MT dynamics in vivo. Future applications of this method may also focus on elucidating cell type and/or compartment-specific MT behaviors, and multicolor correlative imaging of EB1-GFP with other cellular and subcellular markers of interest.&#160;

This study presents a noninvasive intravital neuronal imaging strategy combined with a new software strategy to achieve automated, unbiased tracking and analysis of in vivo microtubule (MT) plus-end dynamics in the sensory dendrites and the neuromuscular junctions of Drosophila.

Microtubules (MTs) play critical roles in neuronal development, but many questions remain about the molecular mechanisms of their regulation and function. ...

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

作者聚焦:了解果蝇神经肌肉接头中的微管网络 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

在体内果蝇前运动神经末端的单分子跟踪

摘要

探索更多视频

此视频中的章节

在果蝇幼虫分部轴突在体内的突触小泡的可视化

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

在完整的果蝇幼虫体内的亚细胞分辨率成像

分析突触调制

一个简单的神经元机械损伤方法学研究

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

发展果蝇神经元的快速丹德林分体动力学的延时实时成像和定量

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

在体内果蝇前运动神经末端的单分子跟踪

摘要

探索更多视频

此视频中的章节

在果蝇幼虫分部轴突在体内的突触小泡的可视化

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

在完整的果蝇幼虫体内的亚细胞分辨率成像

分析突触调制

一个简单的神经元机械损伤方法学研究

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

发展果蝇神经元的快速丹德林分体动力学的延时实时成像和定量

3D Particle Tracking for Noninvasive In Vivo Analysis of Synaptic Microtubule Dynamics in Dendrites and Neuromuscular Junctions of Drosophila

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络