I thank Dr. Maureen Barr, under whose supervision the dauer IL2 remodeling phenotype was initially characterized. Thanks to Dr. C. Britt Carlson for critical reading of the manuscript. Funding in the Barr lab was from the NIH (5R01DK59418) and postdoctoral fellowships from the USDA (2010-65106-20587) and the New Jersey Commission on Spinal Cord Research (CSCR12FEL004). Current funding for this work is from a University of Illinois Urbana-Champaign College of ACES FIRE grant.

1，原油永久性幼虫信息素产生<ol><li>成长OP50 大肠杆菌从大肠杆菌单菌落O 2 / N在37°C，而在100毫升的LB肉汤200rpm振荡。注:此O / N培养可事先保存在4℃。 </li><li>成长氮气C.线虫于6月15日至20日毫米培养皿中以NGM琼脂（见配方在表1）接种50μl的OP50 大肠杆菌的大肠杆菌 ，直到细菌几乎耗尽19。 </li><li>请在1升锥形5X250毫升的媒体中（见配方见表1）烧瓶19。注意:此溶液可预先制成。 </li><li>成长OP50 E.文化大肠杆菌在4升的LB培养基的O / N以1毫升OP50的大肠杆菌接种大肠杆菌 ，并在37℃下振荡16小时。 </li><li>从平板（步骤1.2）与1ml为S基础溶液（见配方见表1）和从4-5板成每4 1升锥形烧瓶中，在步骤1.3中制备的在S媒体转移线虫洗线虫（4- 5板每个瓶）19。 </li><li>离心从步骤1.4的OP50，在650×g离心10分钟，除去上清液。悬浮每个细菌沉淀在10ml为S基础的解决方案。悬浮细菌的等量转移到线虫的每个烧瓶。 </li><li>将培养瓶在定轨摇床上，在室温（20 - 25℃）和100 - 150转4 - 5天，直到溶液开始清除，表明食物的消耗。 </li><li>一旦食物被消耗殆尽，增长OP50 大肠杆菌的另一个O / N培养大肠杆菌在4X 1升LB培养基中。离心细菌，在650×g离心10分钟，并从一个烧瓶中加入沉淀到每一S介质瓶与线虫。返回在S媒体瓶中的轨道摇床中，如在步骤1.7中，额外3-4天，直至食物被再次耗尽。 </li><li>取约1毫升一份的从每个烧瓶中的溶液，并估计线虫浓度由子采样从每1ml等份1微升和计数线虫的数目。注意:线虫种群应该是50,000 - 75,000线虫/毫升。在此浓度下的大多数线虫是dauers。 </li><li>放弃任何污染瓶中。注:污染经常表现为菌丝在步骤1.9所示。 </li><li>线虫离心机在4000 XG在4℃下10分钟。丢弃线虫沉淀，并保留上清液。可选地，看到讨论了使用这些动物做一个低效力的信息素。 </li><li>筛选通过＃1 Whatman过滤纸上清液。注意:保温瓶将加速这一进程。 </li><li>此外，通过过滤0.45微米的无菌真空过滤单元上清。注:滤液可以保持在o / n 4°C或进入1.14。 </li><li>滤液中加入2L烧杯中搅拌棒。放置在加热搅拌台通风柜。煮沸边搅拌，蒸发至约200毫升将溶液转移到500ml烧杯中。继续煮，直到约50ml的乐英尺注:应在该步骤中被视为是可能的解决方案，以沸腾。最后将50ml颜色为黑褐色。在烹调过程中的任何时间，该热可以被关闭，并且将滤液存储的O / N在室温或4℃。 </li><li>放凉后，离心5分钟以1000×克倒出上清液的100毫升的烧杯中，并弃去沉淀。 </li><li>熬上清下降到棕色外壳。从火上移开，并打破了锅铲地壳。添加足够200标准乙醇以覆盖外壳。用封口膜封住烧杯中，并让它坐在O 2 / N在室温。倒掉乙醇到一个干净的烧杯中，并放置到一边。加入新鲜的乙醇覆盖地壳。重复O 2 / N提取3次。池中提取物。 </li><li>使用真空泵干的乙醇提取物。如果真空泵不可用，乙醇提取物添加到一个干净的烧杯和温暖至50°C，而在通风橱中搅拌。注意:一旦乙醇蒸发升渣的粘飞行棕色应该保持。如果加热的情况下，应当小心以不燃烧的溶液，加热时间过长。 </li><li>在10ml的蒸馏水的无菌水溶解残余物。过滤灭菌该溶液用1毫升等分0.22μm的注射器过滤器，并储存在-20℃。注:1ml水来溶解残余物，每100毫升原液体线虫培养物。例如，如果该液体培养烧瓶中的一个由于丢弃污染，则调整所用的水以溶解残余物的量。 </li></ol> 2，确定信息素剂量反应<ol><li>准备永久性幼虫信息素通过使6X5毫升NGM媒体与高贵的琼脂，蛋白胨没有，并补充50微克/毫升硫酸链霉素和一系列永久性幼虫信息素（见配方见表1）4，19，在13×毫米混合成分玻璃培养管，沸腾本生灯，然后倒入切成3厘米的培养皿。 </li><li>称取分icrocentrifuge管。加入1 ml 大肠杆菌的O / N培养的大肠杆菌 OP50到管和离心〜17000 XG的离心管。删除所有的上清液。再次称重该管和确定将细菌沉淀的重量。悬浮用M9缓冲液（见配方在表1）的粒料为4％的最终浓度（ 重量/体积）。等分试样将10μl细菌悬液到每一信息素板4的中心。商店板在o / n RT。 </li><li>蜕皮到成年到信息素板4后接10-15妊娠N2野生型雌雄同体的一天。存储该板在25℃下进行3 - 4小时。 </li><li>摘掉所有的成人雌雄同体的。记录产卵的各板的数量。用封口膜密封板，并恢复至25℃，3天17。 </li><li>算dauers的百分比（ 图2）。注意:对于这两种方法是要注意，重要的是dauers（和较小的前10吨非dauers）会爬上墙壁和到培养皿的盖子。因此重要的是要检查的培养皿为线虫的所有表面。 </li><li>适合数据的剂量-反应曲线并估计欧盟90浓度信息素（足够的信息素诱导90％的动物形成dauers）， 如图3注:我们通常使用逻辑回归模型来估计欧盟90 ，但不同的统计软件包可能会产生类似的估计可供选择的方法。 </li></ol> 3，现场永久性幼虫IL2神经影像学<ol><li>作为协议2使用步骤2.6确定欧盟90值准备永久性幼虫信息素板。 </li><li>诱导使用方法dauers在协议2与应变携带一个综合IL2记者转基因（myIs13 [P KLP-6 :: GFP]，myIs14 [P KLP-6 :: GFP]，myIs16 [P <eM&gt; KLP-6 :: tdTomato]或qIs56 [P 滞后2 :: GFP]）12，20，21注意:当Q Is56时，GFP的表达不会在IL2Qs可见，直到几个小时后永久性幼虫蜕皮和一些初始重塑事件将不可见。另外，虽然从P 滞后2 :: GFP转基因的荧光信号是在IL2s最终明亮永久性幼虫比P KLP-6荧光报告期间，在P 滞后-2 :: GFP转基因也表示弱的头部肌肉的可掩盖细IL2分支机构。 </li><li>下面产卵，取出成人雌雄同体，用封口膜密封板孵育34小时，在25℃17。注:时间为永久性幼虫蜕皮的长度略有不同个体之间。树突树枝状最迅速的时期始于4 - 5小时以下的永久性幼虫蜕皮的发生（39 - 44小时后产蛋在25°C）12。 </li><li>成像的前一天，弥补了10％的溶液（W / V）琼脂糖M9的缓冲区与永久性幼虫信息素在欧盟90浓度。分装约100μl琼脂糖迈上了一个显微镜载玻片和快速，但轻轻地，在上面放置另一张幻灯片创建一个平坦的表面琼脂糖。经过琼脂糖凝固，包裹在保鲜膜和储存的幻灯片在湿度室（用湿纸巾枪头箱）。 </li><li>在34小时的潜伏期后，检查信息素板对已停止抽水咽线虫。单独的幻灯片，使得一个滑动的仅一个表面具有琼脂糖上。选择一个或多个线虫到10％的琼脂糖凝胶垫和1μ升0.1微米的聚苯乙烯珠22。注:使用一个良好的同步人口，多数平板上的线虫会进入蜕皮从L2d的到永久性幼虫。不过，也有可能是其中í变化ndividuals。因此重要的是要注意的附加功能（造口闭塞，咽重构）来估计时间的动物已经在永久性幼虫蜕皮内（见代表性结果为例）的量。 </li><li>确定线虫的方向（左/右，背/腹）。捕获IL2Q树突Z堆叠图像在放大100倍以尽可能低的荧光强度，每15分钟或视需要为特定的实验。继续拍摄图像，直到线虫已经从L2d的角质层分离，固定化是不可能的，或者动物从永久性幼虫恢复（咽开始抽水）。如果不积极成像，放置在一个恒温恒湿试验箱的幻灯片。注:我们使用啶显微镜机动阶段，微分干涉对比（DIC）的光学元件。 </li></ol>

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The authors have nothing to disclose

永久性幼虫特异性神经可塑性和其他永久性幼虫相关的形态学变化的检查需要控制和可靠的形成dauers要么通过基因突变（ 即，DAF-C突变体）或由野生型动物的接触信息素。而使用的基因突变的诱发dauers方便，它可能会混淆的结果。因此，与DAF-C突变收集的数据应该被随后确认与诱导暴露于永久性幼虫信息素进入持久期野生型动物。 的粗永久性幼虫信息素的生产相...

表型可塑性，包括发育和行为的可塑性，可以适应恶劣的环境条件很重要，被认为是动力，发展1。℃。线虫是一种模式生物经常使用的开发和神经生物学研究。在理想情况下，C。在进入成年繁殖期通过四个幼虫阶段线虫的发展。然而，低粮食供应和人口密度高，长的条件下， 线虫可以接受的发育开关，进入一个长寿命和抗逆持久期2。 Dauers显示非dauers几个形态和行为上的差异而可能介导这一抗逆性。的环境线索和遗传途径调节永久性幼虫条目已经广泛地描述3，4，5，6，7，但控制个体的表型变化的分子机制发生ðuring持久形成是不太容易理解。 永久性幼虫特定表型的检测要求生产的永久性幼虫动物。这可以通过以下三种方式:1）从C的饥饿文化采摘线虫 ，2）使用的持久形成组成（DAF-C）突变体，3）诱导持久形成经过纯化信息素。这是比较简单的，从一个标准的NGM板有三挑dauers 线虫的人口已经耗尽了它的细菌的食物供应。在我们手中，一个标准的NGM板最初接种用50μlOP50 大肠杆菌 ，使其生长3天，随后接种3 - 4成人雌雄同体保 ​​持在25℃，将用尽的一个星期内的食物供应和产生几千内的3天dauers（除了众多饥饿非dauers）。然而，这种方法是不令人满意的蜕皮到d中期间出现的审查过程奥尔。因此很难确定哪个几千动物在一个典型的饥饿板的正进入永久性幼虫。此外，使用饿死板提供了可能性进行同步。采用DAF-C突变允许同步和可靠的代dauers的。然而，没有保证特定的daf-C突变不会影响其他永久性幼虫特异性表型单独或与其他突变组合。 à永久性幼虫信息素的存在，最初是由Cassada和罗素暗示，后来被黄金和里德尔证实。该持久性信息素已被化学特点2，8，9，10，纯化信息素是由一个复杂的混合ascarosides，它以不同的形式规范双方的行为和发育过程9,11。使用粗永久性幼虫信息素的提取物可以用于可靠的控制异步同步dauers在野生型背景。 </P&gt; 对神经系统和相关的神经胶质细胞在永久性幼虫幼体形成12，13，14进行改造，其中一些变化是不可逆的，如神经胶质细胞的永久性幼虫13，14在重塑，但其他的重塑事件后，恢复到良好的可逆性环境条件。枝晶树枝状，从单一的树突切换到multidendritic神经元和轴突重塑12:例如，一组4 IL2Q神经元中持久形成包括经过快速，可逆的神经重构。本文的方法允许可靠的体内成像应激诱导的神经可塑性的模式生物。提出了生产和粗永久性幼虫信息素的检测方法被先前描述和编译从几个来源4，8，15，16，17，18。

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:259px;">N2 C. elegans Bristol strain</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td style="width:212px;"></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PT2660 myIs13[Pklp-6::gfp] III</td>
			<td style="width:152px;">Upon request from Schroeder lab</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PT2762 myIs14[Pklp-6::gfp] V</td>
			<td style="width:152px;">Upon request from Schroeder lab</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">JK2868 qIs56[Plag-2::gfp] V</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">OP50 E. coli</td>
			<td style="width:152px;">Caenorhabditis Genetics Center</td>
			<td></td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dishes</td>
			<td style="width:152px;">Tritech Research</td>
			<td>60 mm T3308, 35 mm T3501</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="132">
			<td height="132" style="height:132px;">NGM agar</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">3 g NaCl, 17 g agar, 2.5 g peptone in 975 mL H2O autoclaved cooled to 55&#176;C followed by 1 mL of 5 mg/ml cholesterol in ethanol, 25 mL of 1 M KPO4 buffer (pH 6.0), 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4</td>
		</tr>
		<tr height="356">
			<td height="356" style="height:356px;">S Media</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">S media consists of S basal solution (1.46 g NaCl, 0.25g K2HPO4, 1.5 g KH2PO4, 0.25 ml of 5 mg/ml cholesterol in ethanol and H2O to 250 mL. Autoclaved.) To the S basal solution add 2.5 mL of potassium citrate pH 6.0 (2 g citric acid monohydrate, 29.4 g tri-potassium citrate monohydrate, H2O to 100 mL, autoclaved), 2.5 mL of Trace Metals Solution (1.86 g disodium EDTA, 0.69 g FeSO4&#160;&#8226;7 H2O, 0.2 g MnCl2&#8226;4 H2O, 0.29 g ZnSO4&#160;&#8226;7 H2O, 0.025 g CuSO4&#160;&#8226;5 H2O, H2O to 1 liter. Autoclaved and stored in the dark), 0.75 mL of 1 M CaCl2, and 0.75 mL of 1 M MgSO4.</td>
		</tr>
		<tr height="392">
			<td height="392" style="height:392px;width:259px;">Dauer pheromone plates for dose-response assay</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">Mix 0.085 g Noble agar, 0.5 mL of 0.513 M NaCl and 5 &#181;L of 5 mg/ml cholesterol dissolved in ethanol in each of 6x 15 mL culture tubes. Add sufficient crude pheromone to each tube to create a range of concentrations. Add H2O to bring the volume to 4.725 mL.&#160; Boil over a Bunsen burner until the agar is dissolved and then cool to 55 &#176;C. Add 125 &#181;L 1 M KPO4 buffer (pH 6.0), 50 &#181;l 0.1 M CaCl2, 50 &#181;L 0.1 M MgSO4 and 50 &#181;l of 5 mg/ml streptomycin sulfate to each tube.&#160; Pour the contents of each tube into a 35 mm diameter Petri dish. Let the agar solidify at room temperature overnight (if longer, store in enclosed container to prevent excessive drying).</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microsphere beads 0.1 micron</td>
			<td style="width:152px;">Polysciences Inc.</td>
			<td>00876-15</td>
			<td style="width:251px;"></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">M9 buffer</td>
			<td style="width:152px;">Various</td>
			<td></td>
			<td style="width:251px;">Mix 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 liter. Sterilize by autoclaving</td>
		</tr>
	</tbody>
</table>

in vivo imaging of dauer-specific neuronal remodeling in c. elegans

控制应激诱导的表型可塑性在动物的机制是频繁复杂和困难的体内研究。应力诱发塑性的一个典型的例子是C的持久期线虫 。 Dauers是低浓度的细菌性食物和高浓度永久性幼虫信息素的条件下形成的一种替代发育的幼虫期。 Dauers显示广泛的发育和行为的可塑性。例如，一套四内唇象限（IL2Q）神经元中持久形成进行了广泛可逆的重塑。利用众所周知的环境途径调节永久性幼虫条目，先前建立的方法，用于生产由大型液体线虫培养物粗永久性幼虫信息素被证实。用这种方法，对50000的浓​​度 - 液体培养物75000线虫/ ml的是足以产生一种高效的粗永久性幼虫信息素。粗素效价对射探测器由剂量 - 反应的生物测定rmined。最后，在持久形成用于体内的时间推移的IL2Qs的成像方法进行描述。

生产的粗永久性幼虫信息素产生一种黄色液体（ 图1）即热和冷的稳定。的粗素等分试样储存在-20℃下无限期地与活性无明显损失。下列生产信息素，单剂量 - 反应的生物测定是足够的效力的一个粗略的估计。然而，对于大多数实验是必要的，以重复的生物测定2 - 3次，取平均值，从每个试验。从两个信息素生物试验的代表性结果列于图3中 ，从这些数据的EC 50和...

Watch this Scientific Journal Video about In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans at JoVE.com

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant &#8216;dauer&#8217; juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer.

在永久性幼虫特有的神经重构的C语言活体成像线虫

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of ...

in-vivo-imaging-of-dauer-specific-neuronal-remodeling-in-c-elegans

Research

JoVE Journal

Neuroscience

In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

11.2K 次观看.  University of Illinois Urbana-Champaign. Following exposure to specific environmental stressors, the nematode Caenorhabditis elegans undergoes extensive phenotypic plasticity to enter into a stress-resistant ‘dauer’ juvenile stage. We present methods for the controlled induction and imaging of neuroplasticity during dauer. 

视频: In Vivo Imaging of Dauer-specific Neuronal Remodeling in C. elegans

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

在 C 在体内激光干切断线虫

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Automated Quantification of Synaptic Fluorescence in C. elegans

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall neural circuit function. Synapse strength critically depends on the abundance of neurotransmitter receptors clustered at synaptic sites on the postsynaptic membrane. Receptor levels are established developmentally, and can be altered by receptor trafficking between surface-localized, subsynaptic, and intracellular pools, representing important mechanisms of synaptic plasticity and neuromodulation. Rigorous methods to quantify synaptically-localized neurotransmitter receptor abundance are essential to study synaptic development and plasticity. Fluorescence microscopy is an optimal approach because it preserves spatial information, distinguishing synaptic from non-synaptic pools, and discriminating among receptor populations localized to different types of synapses. The genetic model organism Caenorhabditis elegans is particularly well suited for these studies due to the small size and relative simplicity of its nervous system, its transparency, and the availability of powerful genetic techniques, allowing examination of native synapses in intact animals.
Here we present a method for quantifying fluorescently-labeled synaptic neurotransmitter receptors in C. elegans. Its key feature is the automated identification and analysis of individual synapses in three dimensions in multi-plane confocal microscope output files, tabulating position, volume, fluorescence intensity, and total fluorescence for each synapse. This approach has two principal advantages over manual analysis of z-plane projections of confocal data. First, because every plane of the confocal data set is included, no data are lost through z-plane projection, typically based on pixel intensity averages or maxima. Second, identification of synapses is automated, but can be inspected by the experimenter as the data analysis proceeds, allowing fast and accurate extraction of data from large numbers of synapses. Hundreds to thousands of synapses per sample can easily be obtained, producing large data sets to maximize statistical power. Considerations for preparing C. elegans for analysis, and performing confocal imaging to minimize variability between animals within treatment groups are also discussed. Although developed to analyze C. elegans postsynaptic receptors, this method is generally useful for any type of synaptically-localized protein, or indeed, any fluorescence signal that is localized to discrete clusters, puncta, or organelles.
The procedure is performed in three steps: 1) preparation of samples, 2) confocal imaging, and 3) image analysis. Steps 1 and 2 are specific to C. elegans, while step 3 is generally applicable to any punctate fluorescence signal in confocal micrographs.

Automated Quantification of Synaptic Fluorescence in C. elegans

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

突触荧光自动定量<em&gt; C。线虫</em

Synapse strength refers to the amplitude of postsynaptic responses to presynaptic neurotransmitter release events, and has a major impact on overall ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入:一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and medical applications. Strengths of fluorescence include its sensitivity, specificity, and compatibility with live imaging. Unfortunately, conventional forms of fluorescence microscopy suffer from one major weakness, diffraction-limited resolution in the imaging plane, which hampers studies of structures with dimensions smaller than ~250 nm. Recently, this limitation has been overcome with the introduction of super-resolution fluorescence microscopy techniques, such as photoactivated localization microscopy (PALM). Unlike its conventional counterparts, PALM can produce images with a lateral resolution of tens of nanometers. It is thus now possible to use fluorescence, with its myriad strengths, to elucidate a spectrum of previously inaccessible attributes of cellular structure and organization.
Unfortunately, PALM is not trivial to implement, and successful strategies often must be tailored to the type of system under study. In this article, we show how to implement single-color PALM studies of vesicular structures in fixed, cultured neurons. PALM is ideally suited to the study of vesicles, which have dimensions that typically range from ~50-250 nm. Key steps in our approach include labeling neurons with photoconvertible (green to red) chimeras of vesicle cargo, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a high-resolution PALM image. We also demonstrate the efficacy of our approach by presenting exceptionally well-resolved images of dense-core vesicles (DCVs) in cultured hippocampal neurons, which refute the hypothesis that extrasynaptic trafficking of DCVs is mediated largely by DCV clusters.

We describe how to implement photoactivated localization microscopy (PALM)-based studies of vesicles in fixed, cultured neurons. Key components of our protocol include labeling vesicles with photoconvertible chimeras, collecting sparsely sampled raw images with a super-resolution microscopy system, and processing the raw images to produce a super-resolution image.

神经元的密集核心囊泡的超分辨率成像

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

应用一种适应微流控嗅觉芯片的神经活性成像对雄性线虫神经元的信息素反应

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

Toxicity of misfolded proteins and mitochondrial dysfunction are pivotal factors that promote age-associated functional neuronal decline and neurodegenerative disease across species. Although these neurotoxic challenges have long been considered to be cell-intrinsic, considerable evidence now supports that misfolded human disease proteins originating in one neuron can appear in neighboring cells, a phenomenon proposed to promote pathology spread in human neurodegenerative disease.
C. elegans adult neurons that express aggregating proteins can extrude large (~4 &#181;m) membrane-surrounded vesicles that can include the aggregated protein, mitochondria, and lysosomes. These large vesicles are called &#8220;exophers&#8221; and are distinct from exosomes (which are about 100x smaller and have different biogenesis). Throwing out cellular debris in exophers may occur by a conserved mechanism that constitutes a fundamental, but formerly unrecognized, branch of neuronal proteostasis and mitochondrial quality control, relevant to processes by which aggregates spread in human neurodegenerative diseases.
While exophers have been mostly studied in animals that express high copy transgenic mCherry within touch neurons, these protocols are equally useful in the study of exophergenesis using fluorescently tagged organelles or other proteins of interest in various classes of neurons.
Described here are the physical features of C. elegans exophers, strategies for their detection, identification criteria, optimal timing for quantitation, and animal growth protocols that control for stresses that can modulate exopher production levels. Together, details of protocols outlined here should serve to establish a standard for quantitative analysis of exophers across laboratories. This document seeks to serve as a resource in the field for laboratories seeking to elaborate molecular mechanisms by which exophers are produced and by which exophers are reacted to by neighboring and distant cells.

Quantitative Approaches for Scoring in vivo Neuronal Aggregate and Organelle Extrusion in Large Exopher Vesicles in C. elegans

This protocol describes approaches for detection and quantitation of large aggregate and/or organelle extrusions (~4 &#181;m) produced by C. elegans cells in the form of membrane-bound exophers. We describe strains, growth conditions, scoring criteria, timing, and microscopy considerations needed to facilitate dissection of this debris expulsion mechanism.