This work was supported by grants from the Natural Sciences and Engineering Research Council (NSERC) of Canada and The Canada Foundation for Innovation. Thanks to Dr Paul Mains (University of Calgary, Calgary, Canada) for let-502(sb118ts) strain. Some of the strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

<ol>
	<li>Corsi, A. K., Wightman, B., Chalfie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Transparent+Window+into+Biology:+A+Primer+on+Caenorhabditis+elegans.">A Transparent Window into Biology: A Primer on Caenorhabditis elegans.</a> Genetics. 200, 387-407 (2015).</li><li>Priess, J. R., Hirsh, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+morphogenesis:+the+role+of+the+cytoskeleton+in+elongation+of+the+embryo.">Caenorhabditis elegans morphogenesis: the role of the cytoskeleton in elongation of the embryo.</a> Developmental biology. 117, 156-173 (1986).</li><li>Zhang, H., 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+tension-induced+mechanotransduction+pathway+promotes+epithelial+morphogenesis.">A tension-induced mechanotransduction pathway promotes epithelial morphogenesis.</a> Nature. 471, 99-103 (2011).</li><li>Diogon, M., 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=The+RhoGAP+RGA-2+and+LET-502/ROCK+achieve+a+balance+of+actomyosin-dependent+forces+in+C.+elegans+epidermis+to+control+morphogenesis.">The RhoGAP RGA-2 and LET-502/ROCK achieve a balance of actomyosin-dependent forces in C. elegans epidermis to control morphogenesis.</a> Development. 134, 2469-2479 (2007).</li><li>Gally, C., 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=Myosin+II+regulation+during+C.+elegans+embryonic+elongation:+LET-502/ROCK,+MRCK-1+and+PAK-1,+three+kinases+with+different+roles.">Myosin II regulation during C. elegans embryonic elongation: LET-502/ROCK, MRCK-1 and PAK-1, three kinases with different roles.</a> Development. 136, 3109-3119 (2009).</li><li>Piekny, A. J., Wissmann, A., Mains, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+morphogenesis+in+Caenorhabditis+elegans+integrates+the+activity+of+LET-502+Rho-binding+kinase,+MEL-11+myosin+phosphatase,+DAF-2+insulin+receptor+and+FEM-2+PP2c+phosphatase.">Embryonic morphogenesis in Caenorhabditis elegans integrates the activity of LET-502 Rho-binding kinase, MEL-11 myosin phosphatase, DAF-2 insulin receptor and FEM-2 PP2c phosphatase.</a> Genetics. 156, 1671-1689 (2000).</li><li>Martin, 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=pix-1+controls+early+elongation+in+parallel+with+mel-11+and+let-502+in+Caenorhabditis+elegans.">pix-1 controls early elongation in parallel with mel-11 and let-502 in Caenorhabditis elegans.</a> PloS one. 9, e94684 (2014).</li><li>Sulston, J., Hodgkin, J., Wood, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods. , 587-606 (1988).</li><li>Andersen, E. C., 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+Powerful+New+Quantitative+Genetics+Platform,+Combining+Caenorhabditis+elegans+High-Throughput+Fitness+Assays+with+a+Large+Collection+of+Recombinant+Strains.">A Powerful New Quantitative Genetics Platform, Combining Caenorhabditis elegans High-Throughput Fitness Assays with a Large Collection of Recombinant Strains.</a> G3 (Bethesda). 5, 911-920 (2015).</li><li>Boulier, E. L., Jenna, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+Caenorhabditis+elegans+embryogenesis+using+RNA+interference+and+flow+cytometry.">Genetic dissection of Caenorhabditis elegans embryogenesis using RNA interference and flow cytometry.</a> Methods Mol Biol. 550, 181-194 (2009).</li><li>Verster, A. J., Ramani, A. K., McKay, S. J., Fraser, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+RNAi+screens+in+C.+elegans+and+C.+briggsae+reveal+the+impact+of+developmental+system+drift+on+gene+function.">Comparative RNAi screens in C. elegans and C. briggsae reveal the impact of developmental system drift on gene function.</a> PLoS genetics. 10, e1004077 (2014).</li><li>Ramani, A. K., 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=The+majority+of+animal+genes+are+required+for+wild-type+fitness.">The majority of animal genes are required for wild-type fitness.</a> Cell. 148, 792-802 (2012).</li><li>So, S., Miyahara, K., Ohshima, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+body+size+in+C.+elegans+dependent+on+food+and+insulin/IGF-1+signal.">Control of body size in C. elegans dependent on food and insulin/IGF-1 signal.</a> Genes to cells : devoted to molecular &amp; cellular mechanisms. 16, 639-651 (2011).</li><li>Dineen, A., Gaudet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+signaling+can+act+from+multiple+tissues+to+regulate+C.+elegans+body+size.">TGF-beta signaling can act from multiple tissues to regulate C. elegans body size.</a> BMC developmental biology. 14, 43 (2014).</li><li>Tan, L., Wang, S., Wang, Y., He, M., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bisphenol+A+exposure+accelerated+the+aging+process+in+the+nematode+Caenorhabditis+elegans.">Bisphenol A exposure accelerated the aging process in the nematode Caenorhabditis elegans.</a> Toxicology letters. 235, 75-83 (2015).</li><li>Yu, Z., Yin, D., Deng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combinational+effects+between+sulfonamides+and+metals+on+nematode+Caenorhabditis+elegans.">The combinational effects between sulfonamides and metals on nematode Caenorhabditis elegans.</a> Ecotoxicology and environmental safety. 111, 66-71 (2015).</li></ol>

The authors declare that they have no competing financial interests.

このプロトコルは、胚性の伸びの初期および後期段階を特徴づけるために新たなメトリックを説明しました。 セクション1では、重要なステップは、パッド上の細菌の潜在的な存在です。胚は気密パッドと画像取得時のカバーガラスの間に封入されています。スライドを密閉する2時間以上持続取得中に動物の乾燥を避けるために必要とされます。我々の知る限りでは、ス...

その短いライフサイクル：ほぼ50年間、線虫線虫（Caenorhabditis elegans）が開発、神経生物学、進化、宿主-病原体相互作用などで重要な問題を研究するための強力なモデルとしての地位を確立して1開発の研究では、このモデルの強さはです3日;これらの動物は、遺伝的に改変することができる容易さ。ほとんどが子宮外で生きている動物とその開発における細胞の変位と形態の観察を可能にするその透明。線虫の発達段階は、成人期に続いて、胚形成および4幼虫期（L4へL1）を伴います。胚発生時には、表皮の形態形成は、細胞が、彼らは彼らの接合部を再編成し、それらの個々の形態だけでなく、機能性上皮内での相対的位置を変更する方法を、グループとして移動する方法上皮のより良い理解を可能にする能力についてかなりの注目を集めました。表皮の形態形成は4段階に分かれています。背側表皮細胞の再編成からなる背インターカレーションは、皮下組織と呼ばれます。このように、上皮細胞単層における胚を包む腹側正中線に向かって腹側皮下細胞の遊走になる腹側筐体;初期および後期の伸び虫様幼虫に豆の形胚を変換します。以下の形態形成、胚のハッチとL1幼虫は、その直接の環境で利用可能な細菌を使用して供給し始めます。 胚性の伸びは、したがって、胚発生の後期です。これは、その長手方向軸に沿って胚の伸長と横直径の減少から成ります。これは、皮下細胞の形状の劇的な変更を伴います。伸びが初期および後期段階に分割されています。体壁の筋肉がワットで1.75倍の段階で、収縮開始時に初期の段階ではコンマ段階で開始し、終了しますILD型（wt）胚-非伸長胚と比較して、長さの1.75倍である胚に対応します。その段階で生じる形態形成のプロセスは、主に胚2の前後軸に沿ってその伸びを駆動皮下細胞の先端極に位置するフィラメント状アクチン束（FBS）の収縮によって駆動されます。政府短期の収縮は3キナーゼLET-502 / ROCK、MRCK-1とPAK-1 5によるミオシン軽鎖のリン酸化による制御です。体壁の筋肉が機能するようになると契約を起動したときに、伸びの後期相は、開始されます。これは、背側と腹側の皮下細胞に体壁の筋肉からメカノシグナリングを含み、動物が3を孵化たときに終了します。 伸び欠陥は、一般的に胚のように死んで動物の割合（胚致死性; EMB）を特徴とし、L1幼虫（幼虫逮捕表現型として、その発症を阻止するもの、左心室a）および重量よりも大幅に短くなる。発育停止の段階の同定は、死んだ胚の顕微鏡観察を必要とし、幼虫3-6を逮捕しました 。 最近、このようないくつかのCdc42 / RacのレギュレータとエフェクターPIX-1などの遺伝子、及びPAK-1は、両方の初期および後期伸び3,7の間に形態形成のプロセスを制御することが示されました。また、最近、形態形成のプロセスは早期に伸び、3〜7の間の胚の前後軸に沿って異なることを示しました。これらの知見は、特に初期の伸びの間にそれらの前後軸に沿った胚の形態の特徴付けを可能にする初期または後期の伸長段階およびその他のメトリックをターゲットに新たな評価指標の開発の動機。 これらの新規な方法は、それらの幅ならびに開始時および初期の伸長の終了時に、胚の長さを測定することからなる、彼広告や尾。7 2つのプロトコルはまた、L1ステージ7で同期、新たに孵化した幼虫の長さを測定するために開発されました。 幼虫、成虫および培養培地中に存在する細菌を処理することによって溶解させながら、胚の卵殻は、アルカリ性の次亜塩素酸処理に対してそれらを保護します。この処理は、次に、よく供給成人8の大部分を含む非同期集団から胚を精製するために使用されます。食事制限は、新たに孵化した幼虫を同期させるために使用されます。これらの幼虫の長さを測定し、次いで伸長欠陥を検出するために使用されます。孵化幼虫は、非完全に伸長した胚は、給紙時に「通常の長さ」に回復することができますが、食品の非存在下での逮捕時に彼らの小型化を維持するため、この測定は、培養プレート上で逮捕幼虫の測定よりも好ましいです。 ここでは、ルの測定を可能にする詳細なプロトコルを提示しますタイムラプスDIC顕微鏡と画像解析（プロトコル1）を使用して、胚だけでなく、自分の頭と尾の幅を長くするngth。我々はまた、画像解析（プロトコル2）およびフローサイトメトリー（プロトコル3）を使用して同期幼虫の長さを測定するための詳細なプロトコルを提供します。

<table border="0" cellpadding="0" cellspacing="0" width="696">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agar&#160;</td>
			<td style="width:195px;">BioShop</td>
			<td style="width:119px;">AGR001.500</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:195px;">Bioshop</td>
			<td style="width:119px;">AGA001.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CaCl2 (calcium chloride)</td>
			<td style="width:195px;">Bio Basic Inc.</td>
			<td style="width:119px;">CT1330</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cholesterol</td>
			<td style="width:195px;">Sigma-aldrich</td>
			<td style="width:119px;">C8667</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">cleaning solution&#160;</td>
			<td style="width:195px;">union Biometrica</td>
			<td style="width:119px;">300-5072-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glass coverslips</td>
			<td style="width:195px;">Fisherbrand</td>
			<td style="width:119px;">12-542B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glass slides</td>
			<td style="width:195px;">Fisherbrand</td>
			<td style="width:119px;">12-552-3</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">high fluorescent control particles</td>
			<td style="width:195px;">union Biometrica</td>
			<td style="width:119px;">310-5071-001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">K2HPO4 (potassium phosphate, dibasic)</td>
			<td style="width:195px;">Bio Basic Inc.</td>
			<td style="width:119px;">PB0447</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">KH2PO4 (potassium phosphate, monobasic)</td>
			<td style="width:195px;">Bio Basic Inc.</td>
			<td style="width:119px;">PB0445</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MgSO4 (magnesium sulfate)</td>
			<td style="width:195px;">Sigma-aldrich</td>
			<td style="width:119px;">230391</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Na2HPO4( sodium phosphate, dibasic)</td>
			<td style="width:195px;">Bio Basic Inc.</td>
			<td style="width:119px;">SDB0487</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl (sodium chloride)</td>
			<td style="width:195px;">Bio Basic Inc.</td>
			<td style="width:119px;">DB0483</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pebeo Drawing Gum 45ml</td>
			<td style="width:195px;">p&#233;b&#233;o</td>
			<td>PDG033000</td>
			<td>any art/craft store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peptone</td>
			<td style="width:195px;">BioShop</td>
			<td style="width:119px;">PEP403.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sheath buffer</td>
			<td style="width:195px;">union Biometrica</td>
			<td style="width:119px;">300-5070-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">COPAS Biosort</td>
			<td style="width:195px;">union Biometrica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

頭部、Tail-およびヘッド/テール幅比は、堅牢なメトリックです。 ここで説明するプロトコルは成功し、RhoのGTPアーゼPIX-1のレギュレーターおよびエフェクターの機能を特徴づけるPAK-1とlet-502を</e...

Watch this Scientific Journal Video about Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans at JoVE.com

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

新規メトリックは、線虫の胚伸びを特徴づけるために、<em&gt;線虫（Caenorhabditis elegans）</em

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.

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

novel-metrics-to-characterize-embryonic-elongation-nematode

Research

JoVE Journal

Neuroscience

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

6.0K ビュー.  Université du Québec à Montréal. 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. 

ビデオ: Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（<em&gt; Schistocercaアメリカーナ</em&gt;）嗅覚回路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

神経科学

A Caenorhabditis elegans Model System for Amylopathy Study

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 Alzheimer's disease (AD) and is found in Lewy body dementia, inclusion body myositis and cerebral amyloid angiopathy 1-4. Amylopathies progressively develop with time. For this reason simple organisms with short lifespans may help to elucidate molecular aspects of these conditions. Here, we describe experimental protocols to study A&beta;-mediated neurodegeneration using the worm Caenorhabditis elegans. Thus, we construct transgenic worms by injecting DNA encoding human A&beta;42 into the syncytial gonads of adult hermaphrodites. Transformant lines are stabilized by a mutagenesis-induced integration. Nematodes are age synchronized by collecting and seeding their eggs. The function of neurons expressing A&beta;42 is tested in opportune behavioral assays (chemotaxis assays). Primary neuronal cultures obtained from embryos are used to complement behavioral data and to test the neuroprotective effects of anti-apoptotic compounds.

A Caenorhabditis elegans Model System for Amylopathy Study

We describe methods to study aspects of amylopathies in the worm C. elegans. We show how to construct worms expressing human A&beta;42 in neurons and how to test their function in behavioral assays. We further show how to obtain primary neuronal cultures that can be used for pharmacological testing.

A<em&gt;線虫（Caenorhabditis elegans）</emAmylopathyの研究のための&gt;モデルシステム

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 ...

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.

抗精神病薬の作用のメカニズムを研究するための方法<em&gt;線虫（Caenorhabditis elegans）</em

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

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.

の歩行速度に対するエタノールの効果を測定するためのアッセイ<em&gt;線虫（Caenorhabditis elegans）</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.

の長期カルシウムイメージングのためのアガロースマイクロチャンバー<em&gt;線虫（Caenorhabditis elegans）</em

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

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

ダイナミック定量的感覚検査は、中枢性疼痛処理を特徴づけるために、

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

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.

A<em&gt; Caenorhabditis elegans</em&gt;栄養状態ベースの銅回避アッセイ

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;

線虫の行動で個々 のニューロンのレシオ メトリック カルシウム イメージング

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.

カエノラブディティス・エレガンスにおける樹状突起棘のイメージング

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.

インビボカエノハブディティス・エレガンスニューロンにおける微小管動態と向きの評価

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

新規メトリックは、線虫の胚伸びを特徴づけるために、

要約

さらに動画を探す

この動画の章

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（

A

抗精神病薬の作用のメカニズムを研究するための方法

の歩行速度に対するエタノールの効果を測定するためのアッセイ

の長期カルシウムイメージングのためのアガロースマイクロチャンバー

ダイナミック定量的感覚検査は、中枢性疼痛処理を特徴づけるために、

A

線虫の行動で個々のニューロンのレシオメトリックカルシウムイメージング

カエノラブディティス・エレガンスにおける樹状突起棘のイメージング

インビボカエノハブディティス・エレガンスニューロンにおける微小管動態と向きの評価

新規メトリックは、線虫の胚伸びを特徴づけるために、

要約

さらに動画を探す

この動画の章

ローカスの神経活動を特徴づけるためにマルチユニット記録方法（

A

抗精神病薬の作用のメカニズムを研究するための方法

の歩行速度に対するエタノールの効果を測定するためのアッセイ

の長期カルシウムイメージングのためのアガロースマイクロチャンバー

ダイナミック定量的感覚検査は、中枢性疼痛処理を特徴づけるために、

A

線虫の行動で個々 のニューロンのレシオ メトリック カルシウム イメージング

カエノラブディティス・エレガンスにおける樹状突起棘のイメージング

インビボカエノハブディティス・エレガンスニューロンにおける微小管動態と向きの評価

線虫の行動で個々のニューロンのレシオメトリックカルシウムイメージング