笔者想承认，这项工作是由加拿大自然科学和（NSERC）授予工程研究理事会的支持。

<ol>
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作者什么都没有透露。

本协议描述在胚胎发育中的使用各类显微镜的图像细胞分裂。特别是，该协议强调了如何将图像的神经母细胞的分裂，细胞，可促进表皮形态。后生动物的开发和C在细胞间的沟通对组织形成重要线虫是研究在体内组织形成一个很好的模型。一个事件，很好地描绘组织的相互作用是表皮形态，它涵盖了胚胎一层上皮细胞。特别是，腹侧外壳是其中腹侧表皮细胞迁移到包围胚胎的...

虽然有说明如何像细胞分裂初期C.协议线虫的胚胎，这个协议描述了如何像细胞分裂中期胚胎发育过程中组织内。其中一个在开发过程中的生物成像的主要挑战一直是他们的光毒性的敏感性。然而，增加无障碍旋转盘共聚焦显微镜或扫描显微镜领域已经允许更广泛的成像应用。这两个系统使用固态激光器和散射光，限制紫外线的生物体暴露于的电平。但是，广角台灯仍然可以用于成像在体内 ，尤其是当它们配备相机具有高灵敏度（ 例如 EMCCD），光圈控制和调光（ 例如发光二极管或可调汞灯泡）。本协议描述了如何使用一个基于共聚焦系统或宽视场系统，以图像的细胞分裂中开发C。线虫&lt;/ em&gt;的胚胎。作为一个例子，我们介绍了如何像神经细胞的细胞分裂。神经母细胞可以促进表皮细胞的形态通过提供化学或机械的线索来覆表皮细胞，并提供了细胞间通讯在组织中形成的重要性一个很好的例子。 秀丽隐杆线虫是一种理想的模式生物，由于其透明度和简单的组织机构1显微镜为基础的研究。此外，C。线虫是服从遗传方法和RNAi，并且由于它的许多基因具有人同系物，它可用于鉴定保守机制，组织形成2-5。在C线虫 ，形成表皮层的过程中胚发生时，当胚胎有&gt; 300个细胞。表皮形态包括几个主要阶段，在此期间，胚胎被封闭在一层表皮细胞的收缩和延伸，改造EMBR的哟从一个卵形形状成蜗杆6的细长形状。腹侧外壳描述了这些形态发生的事件之一，当腹侧的表皮细胞向腹侧中线迁移以覆盖胚胎的腹侧表面（ 图1）。首先，两对位于前前缘细胞的迁移对腹中线，在那里他们坚持和保险丝与对侧邻居6。其次是由后部位于口袋的细胞，从而形成楔形形状像创建腹侧口袋6-7的迁移。关闭所述口袋的机制还不是很清楚。一种可能性是，一个supracellular肌动球蛋白收缩结构领带口袋细胞一起在一个荷包喜欢时尚，类似伤口愈合8。有趣的是，一些袖珍细胞的迁移是由相关的神经母细胞9（神经元前体被发现的epidermi下方的特定亚群介导的秒; 图1B）。 以前的研究表明，神经母细胞调节腹侧表皮细胞的迁移和腹侧外壳。VAB-1（肝配蛋白受体）和VAB-2（肝配蛋白配体）高度表达在神经母细胞并彼此促进前部和后部的成神经细胞的分选，并在VAB-1或VAB-2引起腹外壳突变表型10-13。然而，启动子拯救实验表明，VAB-1，也需要在上覆的腹面的表皮细胞和受体的神经细胞分泌其它的指导线索被表达在腹侧表皮细胞9。虽然在任何这些受体的突变可导致腹侧壳体的表型，这是不明确的，如果出现缺陷，由于在成神经细胞的定位或者由于腹侧表皮细胞未能对引导反应的杆14的问题。改变神经母细胞无线网络的划分thout影响其分泌的指导线索的能力可能揭示神经细胞的作用，使她们在表皮形态提供机械输入能力的光。最近，人们发现，一个细胞分裂基因，ANI-1（的Anillin）高度表达在神经母细胞（ 图2A）和其耗尽导致神经细胞分裂缺陷。有趣的是，这些胚胎腹侧显示表型机柜（Fotopoulos，Wernike和Piekny，未发表意见）。 的Anillin是必需的细胞分裂，特别是对于细胞分裂，它描述了该过程，其中一个母细胞物理地划分成两个子细胞。胞质分裂是由肌动球蛋白收缩环，这需要进行严格控制在空间和时间，以确保它是正确加上姐妹染色单体偏析的形成驱动。后生动物胞质分裂的主调节器是RhoA蛋白（ 线虫 RHO-1），小GTP酶是一在莫如其GTP结合形式。环境基金Ect2/ECT-2激活的RhoA，在这之后的RhoA-GTP与形成收缩环并介导其侵入15下游效应相互作用。的Anillin是，通过其C-末端并通过其N-末端肌动蛋白和肌球蛋白结合的RhoA的多结构域蛋白。的Anillin需要稳定在哺乳动物或果蝇S2细胞16的收缩环的位置。的Anillin枯竭导致收缩环经过横向振荡，和胞质最终失败形成多核细胞17-19。有趣的是，尽管C。线虫ANI-1坐标肌动球蛋白收缩在早期胚胎，它不是对胞质分裂至关重要。然而，如上所述，ANI-1是在中胚（Fotopoulos，Wernike和Piekny，未发表的观察结果）所需的成神经细胞的胞质。胞质分裂失败会改变神经细胞的数量和位置，并可能影响到禄化学指导线索振动性，或者它可能会改变组织的机械性能。这两款机型突出对神经母细胞腹侧外壳的非自治作用，组织 - 组织沟通的胚胎发育过程中的重要性。 该实验方案介绍了如何像细胞分裂过程中C.线虫用荧光显微镜中胚。多数实验研究细胞分裂的机制是在内部培养皿（ 例如 ，海拉或S2细胞）的单细胞或早期胚胎细胞与数量有限的（ 如线虫的单细胞胚胎， 非洲爪蟾 ，或棘皮动物进行胚胎）。然而，重要的是要同时组织中研究细胞分裂，因为有外部线索可以影响分割平面的时机和位置。此外，细胞可以提供化学或机械线索来影响邻近组织的发展s，而它，了解细胞间的沟通有助于组织发展过程中，形成很重要。

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			<td height="21" style="height:21px;width:291px;">Agar</td>
			<td style="width:247px;">BioShop Canada Inc.</td>
			<td style="width:185px;">#AGR001.1</td>
			<td style="width:335px;">For making C. elegans NGM and RNAi plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>Bio Basic Inc.</td>
			<td>#9002-18-0</td>
			<td>For making bacteria LB agar plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>BioShop Canada Inc.</td>
			<td>#AGA001.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-mouse Alexa 488 antibody</td>
			<td>Life Technologies Corporation (Invitrogen)</td>
			<td>#A11029</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Anti-mouse anti-GFP antibody</td>
			<td>Roche Applied Science</td>
			<td>#11814460001</td>
			<td></td>
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			<td height="21" style="height:21px;">Anti-rabbit Alexa 568 antibody</td>
			<td>Life Technologies Corporation (Invitrogen)</td>
			<td>#A11011</td>
			<td></td>
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			<td height="21" style="height:21px;">Ampicillin</td>
			<td>BioShop Canada Inc.</td>
			<td>#AMP201.5</td>
			<td>Store powder at 4 &#176;C and dissolved ampicillin at -20 &#176;C</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Bactopetone&#160; (peptone-A)</td>
			<td>Bio Basic Inc.</td>
			<td>#G213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2 (calcium chloride)</td>
			<td>BioShop Canada Inc.</td>
			<td>#C302.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cholesterol</td>
			<td>BioShop Canada Inc.</td>
			<td>#CHL380.25</td>
			<td>Dissolve in ethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAPI&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>#D9542</td>
			<td>Use to stain nucleic acids (DNA)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>BioShop Canada Inc.</td>
			<td>#GLY001.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IPTG (isopropylthio-&#946;-galactoside)</td>
			<td>Bio Basic Inc.</td>
			<td>#367-93-1</td>
			<td>Store powder and dissolved IPTG at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4 (potassium phosphate, monobasic)</td>
			<td>BioShop Canada Inc.</td>
			<td>#PPM666.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">K2HPO4 (potassium phosphate, dibasic)</td>
			<td>BioShop Canada Inc.</td>
			<td>#PPD303.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L4440&#160; (feeding vector)</td>
			<td>Addgene</td>
			<td>#1654</td>
			<td>Keep as glycerol stock at -80 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4&#160;&#160; (magnesium sulfate)</td>
			<td>BioShop Canada Inc.</td>
			<td>#MAG511.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl (sodium chloride)</td>
			<td>Bio Basic Inc.</td>
			<td>#7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4 (sodium phosphate, dibasic)</td>
			<td>Bio Basic Inc.</td>
			<td>#7558-79-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Donkey Serum (NDS)</td>
			<td>Wisent Bioproducts</td>
			<td>#035-110</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">n-propyl 3.4.5-trihydroxybenzoate (propyl gallate)</td>
			<td>Alfa Aesar</td>
			<td>#A10877</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Poly-L-lysine</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>#P8920</td>
			<td>For optimal results coat microscope slides three times&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin</td>
			<td>BioShop Canada Inc.</td>
			<td>#STP101.50</td>
			<td>Store powder at 4 &#176;C and dissolved streptomycin at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetracyclin</td>
			<td>BioShop Canada Inc.</td>
			<td>#TET701.10</td>
			<td>Store powder at 4 &#176;C and dissolved tetracycline at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween-20</td>
			<td>Bio Basic Inc.</td>
			<td>CAS#9005-64-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tryptone</td>
			<td>BioShop Canada Inc.</td>
			<td>#TRP402.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>Bio Basic Inc.</td>
			<td>#8013-01-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing neuroblast cytokinesis during c. elegans embryogenesis

该协议描述了使用荧光显微镜的图像内开发秀丽隐杆线虫的胚胎分裂的细胞。特别是，这个协议的重点在于如何图像划分成神经细胞，这是发现了表皮细胞的下方和可以是用于表皮的形态很重要。组织的形成是后生动物发展是至关重要的，并依赖于外部线索来自邻近组织。C。线虫是一个很好的模式生物来研究组织形态发生在体内 ，由于其透明度和简单的组织，使得其组织容易通过显微镜来研究。腹侧外壳是其中胚胎的腹侧表面被上皮细胞单层的过程。此事件被认为是由底层的神经细胞，它提供化学品的指导线索进行调解覆上皮细胞的迁移提供便利。然而，成神经细胞是高度增殖和也可以充当作为机械基片的腹侧表皮细胞。使用此实验方案的研究可以在组织形成揭开间沟通的重要性，并且可以用来揭示发展组织中参与细胞分裂的基因的作用。

该实验方案描述了C。如何像细胞分裂线虫在胚胎发育中的胚胎。特别地，它描述了如何图象的成神经细胞，其可以促进表皮细胞的形态发生。表皮形态发生是由于表皮细胞形状的变化，迁移和黏附，而且还依赖于化学或机械线索来自相关神经母细胞（ 图1B）的组合。在神经母细胞分泌接收由受体腹表皮细胞，从而调节他们的迁移10,14,25的表面上的指导线索。此外...

Watch this Scientific Journal Video about Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis at JoVE.com

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

本协议描述了如何在图像秀丽隐杆线虫的胚胎组织中分裂的细胞。虽然一些协议描述了如何像细胞分裂在胚胎早期，这个协议描述了如何在图像中胚胎发育晚期一个发展中的组织内的细胞分裂。

可视化的神经细胞胞质分裂过程中<em&gt; C。线虫</em&gt;胚胎

This protocol describes the use of fluorescence microscopy to image dividing cells within developing Caenorhabditis elegans embryos. In particular, this ...

visualizing-neuroblast-cytokinesis-during-c-elegans-embryogenesis

Research

JoVE Journal

Neuroscience

Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

11.7K 次观看.  Concordia University. 本协议描述了如何在图像秀丽隐杆线虫的胚胎组织中分裂的细胞。虽然一些协议描述了如何像细胞分裂在胚胎早期，这个协议描述了如何在图像中胚胎发育晚期一个发展中的组织内的细胞分裂。 

视频: Visualizing Neuroblast Cytokinesis During C. elegans Embryogenesis

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

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

线虫的积极丁酮学习，短期，长期联想记忆检测

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

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 ...

C. elegans Tracking and Behavioral Measurement

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the surface of an agar plate. For the study of the genetic, biochemical, and neuronal basis of behavior, C. elegans is an ideal organism because it is genetically tractable, amenable to microscopy, and shows a number of complex behaviors, including taxis, learning, and social interaction1,2. Behavioral analysis based on tracking the movements of worms as they crawl on agar plates have been particularly useful in the study of sensory behavior3, locomotion4, and general mutational phenotyping5. Our system works by moving the camera and illumination system as the worms crawls on a stationary agar plate, which ensures no mechanical stimulus is transmitted to the worm. Our tracking system is easy to use and includes a semi-automatic calibration feature. A challenge of all video tracking systems is that it generates an enormous amount of data that is intrinsically high dimensional. Our image processing and data analysis programs deal with this challenge by reducing the worms shape into a set of independent components, which comprehensively reconstruct the worms behavior as a function of only 3-4 dimensions6,7. As an example of the process we show that the worm enters and exits its reversal state in a phase specific manner.

C. elegans Tracking and Behavioral Measurement

We have developed a video-rate tracking microscope system that can record and quantify C. elegans behavior at high resolution and high speeds. We have also developed computational methods to reduce the dimensionality of the worm images to a fundamental set of measurements that completely describe the shape of the worm.

线虫跟踪和行为测量

We have developed instrumentation, image processing, and data analysis techniques to quantify the locomotory behavior of C. elegans as it crawls on the ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

海马胰岛素显微注射和<em在体内</em&gt;微透析过程中空间记忆测试

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

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, ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted area of the brain, neural stem cells proliferate and constantly generate neuroblasts that migrate tangentially in chains along the rostral migratory stream (RMS) to reach the olfactory bulb (OB). Once in the OB, neuroblasts switch to radial migration and then differentiate into mature neurons able to incorporate into the preexisting neuronal network. Proper neuroblast migration is a fundamental step in neurogenesis, ensuring the correct functional maturation of newborn neurons. Given the ability of SVZ-derived neuroblasts to target injured areas in the brain, investigating the intracellular mechanisms underlying their motility will not only enhance the understanding of neurogenesis but may also promote the development of neuroregenerative strategies.
This manuscript describes a detailed protocol for the transfection of primary rodent RMS postnatal neuroblasts and the analysis of their motility using a 3D in vitro migration assay recapitulating their mode of migration observed in vivo. Both rat and mouse neuroblasts can be quickly and efficiently transfected via nucleofection with either plasmid DNA, small hairpin (sh)RNA or short interfering (si)RNA oligos targeting genes of interest. To analyze migration, nucleofected cells are reaggregated in &#39;hanging drops&#39; and subsequently embedded in a three-dimensional matrix. Nucleofection per se does not significantly impair the migration of neuroblasts. Pharmacological treatment of nucleofected and reaggregated neuroblasts can also be performed to study the role of signaling pathways involved in neuroblast migration.

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Neuroblast migration is a crucial step in postnatal neurogenesis. The protocol described here can be used to investigate the role of candidate regulators of neuroblast migration by employing DNA/small hairpin RNA (shRNA) nucleofection and a 3D migration assay with neuroblasts isolated from the rodent postnatal rostral migratory stream.

鼠神经母细胞核转染到研究神经细胞迁移<em&gt;在体外</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

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

The mechanisms controlling stress-induced phenotypic plasticity in animals are frequently complex and difficult to study in vivo. A classic example of stress-induced plasticity is the dauer stage of C. elegans. Dauers are an alternative developmental larval stage formed under conditions of low concentrations of bacterial food and high concentrations of a dauer pheromone. Dauers display extensive developmental and behavioral plasticity. For example, a set of four inner-labial quadrant (IL2Q) neurons undergo extensive reversible remodeling during dauer formation. Utilizing the well-known environmental pathways regulating dauer entry, a previously established method for the production of crude dauer pheromone from large-scale liquid nematode cultures is demonstrated. With this method, a concentration of 50,000 - 75,000 nematodes/ml of liquid culture is sufficient to produce a highly potent crude dauer pheromone. The crude pheromone potency is determined by a dose-response bioassay. Finally, the methods used for in vivo time-lapse imaging of the IL2Qs during dauer formation are described.

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 ...