Name: methods for studying the mechanisms of action of antipsychotic drugs in caenorhabditis elegans
Uploaded: 2014-02-04T14:00:00
Description: Watch this Scientific Journal Video about Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans at JoVE.com

这项工作是由美国国立卫生研究院临床科学家发展奖K08NS002083，一个Shervert弗雷泽研究所格兰特和NARSAD青年研究者奖，以埃德加A.巴特纳支持。

1。发展迟缓/致命偷袭含量：野生型（N2）和两个突变（MUT1和MUT2）菌株在12孔板测试三中氯氮平浓度<ol><li>在第1天，倒入2毫升NGM培养基中10到12孔板的各孔中（每孔用直径2厘米），并允许过夜变硬的 ​​长凳上，在室温（RT）。同一天，挑大肠杆菌 OP50细菌菌落，感染了一瓶50毫升LB培养基溶液，它的文化在37℃摇床以220rpm旋转过夜。 </li><li>第2天，转移20微升OP50细菌?...

<ol>
	<li>Donohoe, D. R., Jarvis, R. A., Weeks, K., Aamodt, E. J., Dwyer, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+adaptation+in+C.+elegans+produced+by+antipsychotic+drugs+requires+serotonin+and+is+associated+with+calcium+signaling+and+calcineurin+inhibition.">Behavioral adaptation in C. elegans produced by antipsychotic drugs requires serotonin and is associated with calcium signaling and calcineurin inhibition.</a> Neurosci. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antipsychotic+drugs+disrupt+normal+development+in+Caenorhabditis+elegans+via+additional+mechanisms+besides+dopamine+and+serotonin+receptors.">Antipsychotic drugs disrupt normal development in Caenorhabditis elegans via additional mechanisms besides dopamine and serotonin receptors.</a> Pharmacol. Res. 54, 361-372 (2006).</li><li>Karmacharya, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+effects+of+clozapine:+involvement+of+trace+amine+pathways+in+C.+elegans+and+M.+musculus.">Behavioral effects of clozapine: involvement of trace amine pathways in C. elegans and M. musculus.</a> Brain Res. 1393, 91-99 (2011).</li><li>Saur, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Genome-Wide+RNAi+Screen+in+Caenorhabditis+elegans+Identifies+the+Nicotinic+Acetylcholine+Receptor+Subunit+ACR-7+as+an+Antipsychotic+Drug+Target.">A Genome-Wide RNAi Screen in Caenorhabditis elegans Identifies the Nicotinic Acetylcholine Receptor Subunit ACR-7 as an Antipsychotic Drug Target.</a> PLoS Genet. 9, (2013).</li><li>Avery, L., You, Y. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+developmental+exposure+to+ethanol+on+Caenorhabditis+elegans.">Effects of developmental exposure to ethanol on Caenorhabditis elegans.</a> Alcohol Clin. Exp. Res. 32, 853-867 (2008).</li><li>Kage-Nakadai, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+very+long+chain+fatty+acid+acyl-CoA+synthetase+genes,+acs-20+and+acs-22,+have+roles+in+the+cuticle+surface+barrier+in+Caenorhabditis+elegans.">Two very long chain fatty acid acyl-CoA synthetase genes, acs-20 and acs-22, have roles in the cuticle surface barrier in Caenorhabditis elegans.</a> PLoS One. 5, (2010).</li><li>Partridge, F. A., Tearle, A. W., Gravato-Nobre, M. J., Schafer, W. R., Hodgkin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C.+elegans+glycosyltransferase+BUS-8+has+two+distinct+and+essential+roles+in+epidermal+morphogenesis.">The C. elegans glycosyltransferase BUS-8 has two distinct and essential roles in epidermal morphogenesis.</a> Dev. Biol. 317, 549-559 (2008).</li><li>Leung, C. K., Deonarine, A., Strange, K., Choe, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+and+biosensing+with+fluorescent+C.+elegans+strains.">High-throughput screening and biosensing with fluorescent C. elegans strains.</a> J. Vis. Exp. (51), (2011).</li></ol>

在这里，我们描述了用于测试的APD对C的发展和行为的影响的方法线虫 。 DMSO或乙醇来溶解氯氮平，因为药物是相对不溶于水。因为溶剂已报道影响C.线虫生物学12，DMSO单独或乙醇单独对照组是必不可少的。 DMSO在我们的实验中使用的最高浓度可达3％，这不会对下一个明显的效果线虫的发展。 DMSO可用于许多小分子，或什至一些大的分子， 例如脂氨?...

秀丽隐杆线虫是一种简单的遗传生物经得起大规模的正向和反向遗传筛选和化学遗传筛选。C。线虫是一个广泛的生物活性化合物的光谱敏感，并因此被成功地用于定义各种这样的化合物的作用机制。例如，生物活性物质利用蠕虫药理学包括乙酰胆碱受体激动剂（ 如左旋咪唑，尼古丁，噻烯氢嘧啶，噻嘧啶和），麻醉药（ 如氟烷），咖啡因，胆碱酯酶抑制剂（ 如涕灭威，lannate和敌百虫），氟化物，GABA相关研究化合物（ 如 γ-氨基丁酸和 ​​蝇蕈醇），伊维菌素，百草枯，佛波醇酯和五羟色胺有关的药物（ 如血清素和imiprimine）3。此外，C。线虫已经用于大型的小分子的屏幕，允许发现新的生物活性化合物s和鉴定新基因目标4。 该三线虫基因组包括潜在的抗精神病药物（APD）保守在人类的目标，包括基因编码所需的神经递质的合成和突触的结构和功能5蛋白。因此，C。线虫神经遗传学和神经生物学报价方法发现的APD作用的新的分子机制。线虫，APD的曝光处于发展初期产生发育迟缓，并在较高的浓度，杀伤力2,6。成年期的APD曝光产生的行为表型。例如，氯氮平曝光抑制运动和咽抽水，提高产蛋1,2,7。 APD引起的发育迟缓和杀伤力是强大的表型为大型化工遗传筛选。这些表型是复杂的，只要它们可能有一个以上的细胞和遗传巴斯秒。因此，这样的遗传筛选预期产生的各种间接的药物靶点。然而，我们的实验室已进行候选基因的屏幕和全基因组RNAi筛选为APD引起发育迟缓和杀伤力抑制器，并已成功恢复基因，可能编码直接目标，包括多巴胺，胰岛素和烟碱乙酰胆碱受体2,8。的基础上，成人APD诱导行为遗传筛选也导致了新的APD目标的确定，以及我们现在正在从哺乳动物7两个发育和行为屏幕验证目标。因此，无脊椎动物化学遗传学方法发现的APD作用的新的分子机制似乎是可行5,8。 该三线虫咽的是，包括20个神经元，20肌细胞，和20辅助细胞，包裹由基底膜的器官。类似于哺乳动物的心脏，咽是一个自治第二不断泵食品在来自外部环境的9。咽抽水率抑制妥协食物的摄取，从而突变或药物抑制咽抽水引起发育迟缓或逮捕9。 APD的抑制咽抽水率，占部分为他们的发展和生存能力1,2效果。在这里，我们用非典型APD氯氮平作为一个例子来证明药物检测线虫发育和咽抽水。

<table border="0" cellpadding="0" cellspacing="0" width="406">
	<tbody>
		<tr height="22">
			<td>Clozapine</td>
			<td>Sigma</td>
			<td>C6305</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">D8418</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Acetic acid (HAC)</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:119px;">BP2401</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">Sodium hydroxide(NaOH)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0590-13</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Hypochlorite</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">425044</td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:236px;">Centrifuge 5810</td>
			<td style="width:188px;">Eppendorf</td>
			<td style="width:200px;">5810 000.017</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Incubator shaker</td>
			<td style="width:188px;">New Brunswick Scientific</td>
			<td style="width:200px;">M1246-0006</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:236px;">Low temperature incubator 815</td>
			<td style="width:188px;">Precision Scientific</td>
			<td style="width:200px;">J1790-1B</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:236px;">Stereo microscope</td>
			<td style="width:188px;">Olympus</td>
			<td style="width:200px;">SZX12</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:236px;">Petri dish 35 x 10 mm</td>
			<td style="width:188px;">Fisher Scientific</td>
			<td style="width:200px;">NC9434271</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:236px;">12-well tissue culture plate</td>
			<td style="width:188px;">BD Falcon</td>
			<td style="width:200px;">REF 353043</td>
		</tr>
	</tbody>
</table>

methods for studying the mechanisms of action of antipsychotic drugs in caenorhabditis elegans

秀丽隐杆线虫是一种简单的遗传生物经得起大规模的正向和反向遗传筛选和化学遗传筛选。该三线虫基因组包括潜在的抗精神病药物（APD）保守在人类的目标，包括基因编码所需的神经递质的合成和突触的结构和功能的蛋白质。 APD曝光产生以浓度依赖的方式发育迟缓和/或致死的线虫。这些表型引起的，部分是由APD-诱导抑制咽泵1,2的。因此，发展型有神经肌肉的基础上，使其成为有用的抗精神病药的药理学研究。在这里，我们展示了详细的程序，用于测试对线虫发育和咽抽水APD的影响。对于发育检测，同步胚胎放在线虫生长培养基含有的APD（NGM）板，和ANI发展的阶段MALS，然后每天进行评分。对于咽抽水速率法，上演年轻的成年动物都含有APD的NGM板测试。每单位时间的咽泵的数目被记录下来，并且该泵送率的计算方法。这些测定可用于研究许多其他类型的小分子，甚至大的分子。

1。发育迟缓/杀伤力测定结果一个典型的结果为发育延迟/致死率试验中演示了图1a和1b。当对照组野生型动物已发展到了妊娠成年阶段（ 图1a），暴露于氯氮平野生型动物仍然在年轻的幼虫阶段或已经死了（ 图1b）。图1c显示比较具有代表性的结果抑制基因的突变体（MUT1）和增强子的突变体（MUT2）与野生型在三个?...

Watch this Scientific Journal Video about Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans at JoVE.com

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

方法用于测试的抗精神病药物（APD）提供在秀丽隐杆线虫的效果表现出来。分析描述了用于测试的发展和生存能力和咽抽水率药物作用。这些方法也适用于药理学实验与比APD的其他类药物。

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.  ...

Research

JoVE Journal

Neuroscience

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

科研

神经科学

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

A Caenorhabditis elegans Model System for Amylopathy Study

A Caenorhabditis elegans Model System for Amylopathy Study

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 ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

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Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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

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

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

Agarose Microchambers for Long-term Calcium Imaging 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

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

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

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

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

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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

Imaging Dendritic Spines in Caenorhabditis elegans

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 ...