<p class="jove_content"我们感谢刘爽博士批判性阅读的手稿。 PCL12构造是一个礼物形式克里斯托弗博士D.链接。这项工作是支持由两个国家科学基金会资助（0842708和1026958）和AHA授予FS（09GRNT2250529）。</p

1。转基因蠕虫建设<ol><li>转型。 <ol><li>准备注射垫。热，2％琼脂糖溶解在水中，将下降到玻璃盖玻片上。快速放置第二个下拉盖玻片，轻轻敲击。在琼脂糖固化，滑动盖玻片分开，和烘烤盖玻片垫在真空烘箱中在80℃CO / N。 </li><li>拉移液器。我们使用一个萨特的P-97车夫拉1/0.5毫米OD / ID硼硅毛细血管长丝。移液器都是伪造的，这是打破在稍后阶段开放与闭合尖端。 </li><li>准备注射组合。使注射含有感兴趣的DNA（20毫微克/微升每构造）附加空质粒DNA的终浓度为150纳克/微升。离心注射液混合，以消除任何污染物。这一步是至关重要的，因为污染物降低转化效率。 5微升（碎片和其他污染物在底部收集）转移到一个新的管到bË用于注射。 </li><li>注射混合装入毛细。的移液管的底部被浸入在注射的组合。通常，0.5μL足以注入100蠕虫。 </li><li>装入注射针。吸管插入到显微持有人，并打破它打开安装在玻片上，或者对琼脂糖凝胶垫碎片盖玻片边缘摩擦笔尖。这是一个关键的一步，过大的秘诀损害蠕虫和太小的提示很容易堵塞。破碎小费的质量可以判断的形状和最重要的是由流量。评估的流率可以从打开的前端浸渍在卤化碳700油中的注射压力的下降流动的气泡的大小。 </li><li>蠕虫传输到注射垫。将下降700卤烃油注射垫和几个（1或2，适合初学者）蠕虫转移到油。使用蠕虫挑推蠕虫垫，直到上Ÿ坚持琼脂糖。蠕虫的方向应该与腹侧朝向相同的方向中的行。避免接触蠕虫的头部。如果多次尝试后未能坚持蠕虫垫，更换新鲜垫或增加琼脂糖厚度和/或浓度。 </li><li>将吸管插入该蠕虫。通过移动舞台，蠕虫定位下的吸管。核心性腺吸管的位置，因为它的许多生殖细胞的细胞核细胞质共享。这增加了注射的DNA的许多后代的可能性。移液管应位于几乎平行于蜗杆（约15°-25°的角度）。垂直推冰山的吸管跌蠕虫的身体，直到皮肤被压下。然后轻轻拍打操纵器上，诱使尖端的插入。为了达到最佳效果，同时注入性腺。 </li><li>注入的DNA溶液。施加压力，以吸管，直到性腺肿。停止流和关闭吸管拉​​蠕虫。测试针流量，然后将到下一个蜗杆和重复注射的步骤。 </li><li>恢复蠕虫：加一滴（约10微升）恢复缓冲蠕虫和孵化，直到蠕虫开始游泳。加入等体积的M9，等到恢复蠕虫游泳和重复几次，直到解决方案主要是M9。个别蠕虫种子板块。 </li></ol></li><li>集成。 <ol><li>到一个新的板块，将40名健康，良好的喂养，L4蠕虫</li><li> 4000拉德γ-射线照射40分钟。照射蠕虫（P0）传送新鲜，OP50种子板（4蠕虫/板）。蠕虫也可以照射剂量为300焦耳/米2的UV。 </li><li>从各板块个别板块转移10-20 F1转化，标示板与相应的P0起源。 </li><li>单出2-4 F2转化为每个F1分开板。检查F3后代100％传输改造标记。通常情况下，后代1-3％一个照射的蠕虫WILl具有整合事件。 </li><li>股票中的三个或更多个独立的转化体线。 </li></ol></li></ol> 2。行为分析<ol><li>年龄同步。 <ol><li>在标准的10厘米NGM板+ OP50 E.成长蠕虫妊娠成人一个人口众多的大肠杆菌，直到达到（3-5天）。 </li><li>收集在50毫升猎鹰管蠕虫悬浮于1毫升M9的缓冲区。 </li><li>在450×g离心3分钟，加入5毫升M9缓冲液，离心。弃上清。重复3至4倍。 </li><li>在年底的最后离心，去除上清，加入颗粒蠕虫的基本次氯酸钠溶液（0.5 M氢氧化钠，1％次氯酸钠新鲜混合）10卷。在室温下孵育约10分钟。该裂解反应可以放置在盖玻片上的裂解反应的下降，并在显微镜下检查蠕虫监测。当大约80％的蠕虫被打破了反应，应停止。 </li><li>停止插件裂解反应克蛋无菌缓冲液相同的体积。 </li><li>在450×g离心5分钟，通过离心收集鸡蛋（尸体）。 </li><li>鸡蛋洗净，用无菌鸡蛋缓冲区2-3X。 </li><li> M9的缓冲区和种子NGM板标准孵蛋O / N。 </li></ol></li><li>趋化实验。 <ol><li>准备几件琼脂0.5x0.5x0.5厘米左右。我们存入琼脂在10厘米的板，并从那里，我们削减了琼脂块。 </li><li>浸泡琼脂块在溶液中含有所需的引诱剂（在饱和附近的饱和浓度）2小时。典型的引诱是赖氨酸（0.5米），生物素（0.2米）。 </li><li>定金琼脂在10厘米的试验板，其中一个测试点的位置，以及控制点（ 图1A）已经被标记块。允许平衡，形成梯度O / N（ 图1B）。一个单一的实验准备5大板块。 </li><li>之前的实验中加10微升20毫米楠3（ANESThetic）对各点。 </li><li>将20岁的同步蠕虫在板的中心。在孵化器在20℃下将板</li><li> 1小时后，算上动物测试/控制点和趋化指数（CI）计算如下： <img alt="公式1" fo:content-width=".863in" fo:src="/files/ftp_upload/50435/50435eq1highres.jpg" src="/files/ftp_upload/50435/50435eq1.jpg" />其中，N 测试和N Cnt的，表明动物的总数，在测试点的动物的数量，在控制点的动物的数量。 </li></ol></li></ol> 3。初级胚胎细胞培养<ol><li>裂解蠕虫所描述的年龄同步。 </li><li>停止的裂解反应，在450×g离心5分钟，加入无菌鸡蛋相同体积的缓冲液和离心。轻轻弃上清小心不要失去颗粒鸡蛋。重复2 - 3倍，或直至上清明确。 </li><li>重悬浮颗粒鸡蛋（和尸体）在2毫升无菌蛋缓冲液，并加入2毫升的蛋缓冲液的无菌60％的蔗糖。混合解决方案，直到鸡蛋完全悬浮（离心作用下，他们往往会形成团块）由专人或以涡旋。 </li><li>以450×g离心15分钟。 </li><li>小心转移上清（含鸡蛋）的无菌试管中。丢弃颗粒包含屠体及其他副产品裂解。 </li><li>蛋的蛋缓冲液再悬浮和离心分离，在450×g离心5分钟，以除去残留的蔗糖。轻轻收集，弃上清。重复3倍。 </li><li>重悬沉淀层流罩下无菌鸡蛋缓冲液含1 U / ml的几丁质酶RT消化蛋壳的鸡蛋。 30分钟后开始监测反应（倒细胞培养显微镜下）。每一批次的几丁质酶有一个稍微不同的活动。通常在1小时内消化完毕。 </li><li>大约有70-80％的蛋壳是由几丁质酶添加CM-15（L-15细胞培养MED消化鎓含有10％胎牛血清，50单位/ ml青霉素和50μg/ ml的链霉素）。游离细胞使用注射器用27号。过滤5.0微米的过滤器，取出完整的胚胎细胞和幼虫，一丛丛的细胞悬液。 </li><li>颗粒解离的细胞悬浮液通过离心分离，在450×g离心15分钟。取出上清，悬浮颗粒在CM-15细胞培养基。 </li><li>板分离的细胞上的玻璃盖通知单预先涂有花生凝集素（0.1毫克/毫升）溶解在水：细胞必须坚持到基板中，为了区别。 </li><li>细胞可以在室温下（16-20℃）在空气中保持2周以上。 </li></ol>

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作者宣称，他们有没有竞争经济利益。

在这里，我们描述相结合的办法，来研究细胞和分子方面，用C amylopathies 线虫 。这种方法的优点包括：1）成本低。 线虫保持在正常与细菌接种的陪替氏培养皿中，在室温下。 2）强大的遗传学。转基因动物在几个月内，可以得到各种各样的启动子序列可用来驱动在特定神经元的所需基因的表达。 3）操作简单，良好的特点，神经系统。C.线虫拥有一个非常简单的神经系?...

β淀粉样蛋白（Aβ）是36-43个氨基酸的肽的顺序裂解后形成的淀粉样前体蛋白（APP），β和γ分泌1。的γ分泌的Aβ肽的C-末端进行处理，并负责可变长度5。 Aβ的最常见的形式是Aβ40和Aβ42，后者更常见的病理条件下，如AD 5相关联。 Aβ形式在高浓度的β-折叠，聚集形成淀粉样纤维6。存款的原纤维周围神经元的老年斑的主要成分。两个斑块和斑块扩散，非Aβ寡聚体，被认为构成了潜在的致病形式的Aβ。 实验室神经元amylopathies研究是复杂的事实，这些条件随时间的进展。因此，它是重要的吨至发展互补基因听话的动物模型小鼠寿命短。这些模型可以用来阐明具体方面amylopathies典型的细胞和分子，凭借其简单，有利于把握问题的实质。 线虫瀑布是这一类。它有一个很短的寿命，〜20天，除了基本的细胞过程，包括调节基因表达，蛋白质贩运，神经元连接，突触，细胞信号传导，和死亡的哺乳动物7类似。蠕虫的独特功能包括强大的遗传学和缺乏的血管系统，使独立研究神经元损伤血管损伤。另一方面，人脑的缺乏限制下使用线虫研究神经退行性疾病的许多方面。此外，病变的解剖分布的再现和识别不能进行这种微生物。其他限制ations包括两个基因表达谱的差异和复杂的行为和记忆功能的减值难以评估。在这里，我们描述的方法来生成C。线虫模型amylopathies。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>1. NGM</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>3 g</td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>AMRESCO</td>
			<td>J637</td>
			<td>17 g</td>
		</tr>
		<tr>
			<td>Bacto-peptone</td>
			<td>AMRESCO</td>
			<td>J636</td>
			<td>2.5 g</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 975 ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving, then add the following items and mix well</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Sigma-Aldrich</td>
			<td>C3045</td>
			<td>1 ml of 5 mg/ml stock( in ethanol)</td>
		</tr>
		<tr>
			<td>Potassium phosphate buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>25 ml of 1M stock</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>2. Potassium phosphate buffer</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>108.3 g</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>P3786</td>
			<td>35.6 g</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>3. M9 buffer</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P5655</td>
			<td>3 g</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S5136</td>
			<td>6 g</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Magnesium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>M2643</td>
			<td>1 ml of 1 M stock</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>4. Egg buffer (pH 7.3, 340 mOsm)</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td>118 mM</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405</td>
			<td>48 mM</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C5670</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Magnesium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>M4880</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310</td>
			<td>25 mM</td>
		</tr>
		<tr>
			<td>Distilled Water</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Bring to 1 L</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Sterilized by autoclaving</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5. CM-15</td>
		</tr>
		<tr>
			<td>L-15 culture medium</td>
			<td>Gibco</td>
			<td>11415</td>
			<td>450 ml</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td>50 ml</td>
		</tr>
		<tr>
			<td>Penicillin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>50 units/ml</td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td>50 g/ml</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Adjust to 340 mOsm with sucrose then sterile filter into autoclaved bottles and store at 4 &deg;C</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>6. Other Reagents</td>
		</tr>
		<tr>
			<td>Halocarbon 700 oil</td>
			<td>Halocarbon Products</td>
			<td>9002-83-9</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 &mu;m Acrodisc Syringe Filter</td>
			<td>PALL Co.</td>
			<td>4199</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Chitinase</td>
			<td>Sigma-Aldrich</td>
			<td>C6137-5UN</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lectin (peanut)</td>
			<td>Sigma-Aldrich</td>
			<td>L0881-10MG</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S320</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lysine</td>
			<td>Sigma-Aldrich</td>
			<td>L5501</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Biotin</td>
			<td>Sigma-Aldrich</td>
			<td>B4639</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium hypochlorite solution</td>
			<td>Sigma-Aldrich</td>
			<td>425044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma-Aldrich</td>
			<td>71289</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

a caenorhabditis elegans model system for amylopathy study

的Amylopathy是一个术语，描述了异常的β淀粉样蛋白（Aβ）的合成和积累组织中随着时间的推移。 Aβ是阿耳茨海默氏病（AD）的一个标志，并且被发现在路易体痴呆，包涵体肌炎和脑淀粉样血管病1-4。 Amylopathies随着时间的推移逐步发展。出于这个原因，简单的有机体寿命短，可能有助于阐明分子方面的这些条件。在这里，我们描述了实验协议利用线虫研究Aβ介导的神经退行性疾病。因此，我们构建转基因蠕虫通过注射DNA编码人类Aβ42到成年雌雄同体的合胞性腺。转化线是稳定的诱变诱发的整合。线虫是年龄同步收集和播种他们的鸡蛋。测试时机行为分析的功能神经元表达Aβ42（趋化实验S）。从胚胎的原代神经元培养物的使用行为数据的补充和测试抗凋亡的化合物的神​​经保护作用。

我们的协议中，我们研究了人类Aβ42低聚物对神经元功能8。推出了平均1两端的限制性内切酶的引物扩增片段编码人类Aβ42和序列消防载体pPD50.52的人工信号肽编码从结构PCL12 9使用。然后将该片段插入到含有2481 bp的FLP-6启动子序列的一个构造在pPD95.75火焰矢量之间的独特的SMA 1站点10。使用协议1中描述的改造技术，我们构建了转基因蠕虫表达Aβ42</su...

Watch this Scientific Journal Video about A Caenorhabditis elegans Model System for Amylopathy Study at JoVE.com

A Caenorhabditis elegans Model System for Amylopathy Study

我们描述的方法来研究蠕虫amylopathies方面<em&gt; C。线虫</em&gt;。我们将展示如何构建表达Aβ的蠕虫<sub&gt; 42</sub&gt;在神经元和如何测试其功能行为分析。进一步的研究表明如何获取药理试验，可用于主培养的神经元。

一<em&gt;线虫</em&gt;研究Amylopathy模型系统

Amylopathy is a term that describes abnormal synthesis and accumulation of amyloid beta (A&beta;) in tissues with time. A&beta; is a hallmark of ...

a-caenorhabditis-elegans-model-system-for-amylopathy-study

Research

JoVE Journal

Neuroscience

A Caenorhabditis elegans Model System for Amylopathy Study

10.4K 次观看.  University of Medicine and Dentistry of New Jersey. 我们描述的方法来研究蠕虫amylopathies方面 C。线虫。我们将展示如何构建表达Aβ的蠕虫 42在神经元和如何测试其功能行为分析。进一步的研究表明如何获取药理试验，可用于主培养的神经元。

视频: A Caenorhabditis elegans Model System for Amylopathy Study

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &amp;#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

含量测定β-淀粉样蛋白的毒性，使用转基因 C。线虫模型

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

科研

神经科学

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

方法研究抗精神病药物的作用机制在<em&gt;秀丽隐杆线虫</em

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the development of more effective treatments is the relative lack of understanding of the molecular underpinnings of the effects of ethanol on behavior. The nematode, Caenorhabditis elegans (C.&#160;elegans), provides a useful model in which to generate and test hypotheses about the molecular effects of ethanol. Here, we describe an assay that has been developed and used to examine the roles of particular genes and environmental factors in behavioral responses to ethanol, in which locomotion is the behavioral output. Ethanol dose-dependently causes an acute depression of crawling on an agar surface. The effects are dynamic; animals exposed to a high concentration demonstrate an initial strong depression of crawling, referred to here as initial sensitivity, and then partially recover locomotion speed despite the continued presence of the drug. This ethanol-induced behavioral plasticity is referred to here as the development of acute functional tolerance. This assay has been used to demonstrate that these two phenotypes are distinct and genetically separable. The straightforward locomotion assay described here is suitable for examining the effects of both genetic and environmental manipulations on these acute behavioral responses to ethanol in C.&#160;elegans.

An Assay for Measuring the Effects of Ethanol on the Locomotion Speed of Caenorhabditis elegans

C. elegans is a useful model for studying the effects of ethanol on behavior. We present a behavioral assay that quantifies the effects of ethanol on the locomotion speed of crawling worms; both initial sensitivity and the development of acute functional tolerance to ethanol can be measured with this assay.

测量乙醇的影响上的步态速度的测定<em&gt;秀丽隐杆线虫</em

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. Embryonic elongation of the nematode Caenorhabditis elegans is a late developmental stage consisting of the elongation of the embryo along its longitudinal axis. This developmental stage is controlled by intercellular communication between hypodermal cells and underlying body-wall muscles. These signaling mechanisms control the morphology of hypodermal cells by remodeling the cytoskeleton and the cell-cell junctions. Measurement of embryonic lethality and developmental arrest at larval stages as well as alteration of cytoskeleton and cell-cell adhesion structures in hypodermal and muscle cells are classical phenotypes that have been used for more than 25 years to dissect these signaling pathways. Recent studies required the development of novel metrics specifically targeting either early or late elongation and characterizing morphogenic defects along the antero-posterior axis of the embryo. Here, we provide detailed protocols enabling the accurate measurement of the length and the width of the elongating embryos as well as the length of synchronized larvae. These methods constitute useful tools to identify genes controlling elongation, to assess whether these genes control both early and late phases of this stage and are required evenly along the antero-posterior axis of the embryo.

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Here we detail protocols specifically designed to monitor morphogenic defects that occur during early and late phases of embryonic elongation of the nematode Caenorhabditis elegans. Ultimately, these protocols are designed to identify genes that regulate these phases and to characterize their differential requirements along the antero-posterior axis of the embryo.

新颖的指标来表征线虫胚胎伸长率<em&gt;秀丽隐杆线虫</em

Dissecting the signaling pathways that control the alteration of morphogenic processes during embryonic development requires robust and sensitive metrics. ...

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state.
We have developed an assay that measures a nematode&#39;s ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated.
Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

Here, we present a Caenorhabditis elegans-specific assay designed to evaluate changes in copper aversion behavior and the ability to locate a common food source, as the organism progresses from a well-fed to starved nutritional state.

一个<em秀丽隐杆线虫</em基于营养状态的铜反转测定

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

比率钙显像对单个神经元的行为线虫线虫

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic spines have been described for DD GABAergic neurons as the input sites from presynaptic cholinergic neurons in the motor circuit of Caenorhabditis elegans. This synaptic circuit can now serve as a powerful new in vivo model of spine morphogenesis and function that exploits the facile genetics and ready accessibility of C. elegans to live-cell imaging. 
This protocol describes experimental strategies for assessing DD spine structure and function. In this approach, a super-resolution imaging strategy is used to visualize the intricate shapes of actin-rich dendritic spines. To evaluate the DD spine function, the light-activated opsin, Chrimson, stimulates the presynaptic cholinergic neurons, and the calcium indicator, GCaMP, reports the evoked calcium transients in postsynaptic DD spines. Together, these methods comprise powerful approaches for identifying genetic determinants of dendritic spines in C. elegans that could also direct spine morphogenesis and function in the brain.

Imaging Dendritic Spines in Caenorhabditis elegans

Dendritic spines are important cellular features of the nervous system. Here live imaging methods are described for assessing the structure and function of dendritic spines in C. elegans.&#160;These approaches support the development of mutant screens for genes that define dendritic spine shape or function.

秀丽隐杆线虫树突棘成像

Dendritic spines are specialized sites of synaptic innervation modulated by activity and serve as substrates for learning and memory. Recently, dendritic ...

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

在体内卡诺哈布德炎电子神经元微管动力学和方向评估

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Methods Article

一