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 border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">PDMS (Sylgard 184 silicone elastomer kit)</td>
			<td style="width:163px;">Dow Corning Corporation</td>
			<td colspan="2" style="width:253px;">000000000001064291</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass-bottomed Culture Dish</td>
			<td>In Vitro Scientific</td>
			<td>D29-20-1.5-N</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Optical Stereo-microscope</td>
			<td>Olympus</td>
			<td>SZ51</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Curved Tweezers (Style 5/45)</td>
			<td>Electron Microscopy Sciences</td>
			<td>0208-5AC-PO</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Minutien Pins (0.1mm)</td>
			<td>Fine Science Tools</td>
			<td>26002-10</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Schneider&#39;s Insect Medium</td>
			<td>Sigma, Life Sciences</td>
			<td>S0146</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Vannas Spring Scissors</td>
			<td>Fine Science Tools</td>
			<td>15000-00</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fine Forceps (Dumont #5)</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ELYRA PS.1 Microscope</td>
			<td>Zeiss</td>
			<td>PS.1</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phosphate Buffered Saline (PBS)</td>
			<td>Lonza</td>
			<td>17-515Q</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde (16%)</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T-9284</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Normal Goat Serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Maxyclear snaplock microcentrifuge Tube (1.7 mL)</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Zen Black acquisition software</td>
			<td>Carl Zeiss, 2012</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Metamorph software</td>
			<td style="width:163px;">Molecular Devices</td>
			<td style="width:87px;">7.7.8</td>
			<td>PALM Tracer Plugin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fly strain</td>
			<td></td>
			<td colspan="2">w1118; HA-Sx1A-mEos2</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

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

科研

神经科学

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 ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

膜片钳电容测量和Ca 2 +单神经末梢成像在视网膜切片

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of primary afferent fibers mediated by GABAergic axo-axonal synapses (primary afferent depolarization). The strength of primary afferent depolarization can be measured by recording of volume-conducted potentials at the dorsal root (dorsal root potentials, DRP). Pathological changes of presynaptic inhibition are crucial in the abnormal central processing of certain pain conditions and in some disorders of motor hyperexcitability. Here, we describe a method of recording DRP in vivo in mice. The preparation of spinal cord dorsal roots in the anesthetized animal and the recording procedure using suction electrodes are explained. This method allows measuring GABAergic DRP and thereby estimating spinal presynaptic inhibition in the living mouse. In combination with transgenic mouse models, DRP recording may serve as a powerful tool to investigate disease-associated spinal pathophysiology. In vivo recording has several advantages compared to ex vivo isolated spinal cord preparations, e.g. the possibility of simultaneous recording or manipulation of supraspinal networks and induction of DRP by stimulation of peripheral nerves.

Measuring Spinal Presynaptic Inhibition in Mice By Dorsal Root Potential Recording In Vivo

GABAergic presynaptic inhibition is a powerful inhibitory mechanism in the spinal cord important for motor and sensory signal integration in spinal cord networks. Underlying primary afferent depolarization can be measured by recording of dorsal root potentials (DRP). Here we demonstrate a method of in vivo recording of DRP in mice.

测量脊髓突触前抑制小鼠背根电位记录<em&gt;在体内</em

Presynaptic inhibition is one of the most powerful inhibitory mechanisms in the spinal cord. The underlying physiological mechanism is a depolarization of ...

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic pathways. Quantitative descriptions are rarely performed, although genetic manipulations produce a range of phenotypic effects and variations are observed even among individuals within control groups.&#160;Emerging evidence shows that morphology, size and location of organelles play a previously underappreciated, yet fundamental role in cell function and survival. Here we provide step-by-step instructions for performing quantitative analyses of phenotypes at the Drosophila larval neuromuscular junction (NMJ). We use several reliable immuno-histochemical markers combined with bio-imaging techniques and morphometric analyses to examine the effects of genetic mutations on specific cellular processes. In particular, we focus on the quantitative analysis of phenotypes affecting morphology, size and position of nuclei within the striated muscles of Drosophila larvae. The Drosophila larval NMJ is a valuable experimental model to investigate the molecular mechanisms underlying the structure and the function of the neuromuscular system, both in health and disease. However, the methodologies we describe here can be extended to other systems as well.

Why Quantification Matters: Characterization of Phenotypes at the Drosophila Larval Neuromuscular Junction

Morphology, size and location of intracellular organelles are evolutionarily conserved and appear to directly affect their function. Understanding the molecular mechanisms underlying these processes has become an important goal of modern biology. Here we show how these studies can be facilitated by the application of quantitative techniques.

为什么量化事项:表型的表征在<em&gt;果蝇</em&gt;幼虫神经肌肉接头

Most studies on morphogenesis rely on qualitative descriptions of how anatomical traits are affected by the disruption of specific genes and genetic ...

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles in the skin of the mouse pinna. The preparation is simple and relatively fast, reliably yielding extensive regions of multiple labeled units of living nerve terminals to study uptake and release of styryl pyridinium dyes extensively used in studies of vesicle recycling. Subdividing the preparations before labeling allows test vs. control comparisons in the same ear from a single individual. Helpful tips are given for improving the quality of the preparation, the labeling and the imaging parameters. This new system is suitable for assaying pharmacologically and mechanically-induced uptake and release of these vital dyes in lanceolate terminals in both wild-type and genetically modified animals. Examples of modulatory influences on labeling intensity are given.

Optical Monitoring of Living Nerve Terminal Labeling in Hair Follicle Lanceolate Endings of the Ex Vivo Mouse Ear Skin

Here we describe a novel preparation for imaging live lanceolate sensory terminals of palisade endings that innervate mouse ear skin hair follicles during staining and destaining with styryl pyridinium dyes.

在毛囊披针形结局生活神经末端标记的光学监测<em&gt;离体</em&gt;鼠耳皮肤

A novel dissection and recording technique is described for optical monitoring staining and de-staining of lanceolate terminals surrounding hair follicles ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

通过计算机跟踪检测大鼠嗅觉行为<em&gt;果蝇</em&gt;在四象限嗅

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

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 ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons extend growth cones to navigate along specific pathways by responding to a large number of axon guidance cues that emanate from the surrounding extracellular environment. Growth cone target recognition also plays a critical role in neuromuscular specificity. This work presents a standard immunohistochemistry protocol to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. This protocol includes a few key steps, including a genotyping procedure, to sort the desired mutant embryos; an immunostaining procedure, to tag embryos with fasciclin II (FasII) antibody; and a dissection procedure, to generate filleted preparations from fixed embryos. Motor axon projections and muscle patterns in the periphery are much better visualized in flat preparations of filleted embryos than in whole-mount embryos. Therefore, the filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition, and it can also be applied to both loss-of-function and gain-of-function genetic screens.

Visualization of the Axonal Projection Pattern of Embryonic Motor Neurons in Drosophila

This work details a standard immunohistochemistry method to visualize motor neuron projections of late stage-16 Drosophila melanogaster embryos. The filleted preparation of fixed embryos stained with FasII antibody provides a powerful tool to characterize the genes required for motor axon pathfinding and target recognition during neural development.

胚胎运动神经元轴突投影模式的可视化<em&gt;果蝇</em

The establishment of functional neuromuscular circuits relies on precise connections between developing motor axons and target muscles. Motor neurons ...