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.

פרוטוקול זה מתאר מדדי רומן לאפיין מוקדם שלבים מאוחר של התארכות עוברית. בסעיף 1, השלב הקריטי הוא הימצאות אפשרית של חיידקים על כן השיגור. העוברים מוקפים הרמטית בין המשטח ואת coverslip במהלך רכישת תמונה. איטום המגלשה נדרש להימנע התייבשות...

במשך כמעט 50 שנים elegans Caenorhabditis נמטודות ביססה את עצמה כמודל רב עוצמה כדי לחקור שאלות חשובות בפיתוח, נוירוביולוגיה, אבולוציה, אינטראקציות הפתוגן המארח, וכו &#39;1 עוצמת המודל הזה בחקר התפתחות טמון: מחזור החיים הקצר שלה של 3 ימים; הקלות שבה החיות האלה ניתן לשנות גנטיות; השקיפות שלה המאפשר התצפית של עקירת תא ומורפולוגיה בחיות חיים והתפתחותה כי הוא בעיקר רחמית נוספת. שלבי ההתפתחות של נמטודות לערב עובר וארבעה שלבי זחל (L1 כדי L4), ואחריו לבגרות. במהלך התפתחות עוברית, morphogenesis אפידרמיס משך תשומת לב רבה ביכולתה לאפשר הבנה טובה יותר של איך אפיתל תאי נודדים כקבוצה, איך הוא מחדש צומת שלהם ולשנות המורפולוגיה האישית שלהם, כמו גם המיקום היחסי שלהם בתוך האפיתל פונקציונלי.morphogenesis עוריות מחולק לארבעה שלבים: עיבור הגבה מורכב בארגון מחדש של תאי אפידרמיס הגבה, המכונה hypodermis; מתחם גחון, מורכב נדידת תאי hypodermal גחון כלפי קו אמצע הגחון ובכך עוטף את העובר בתוך monolayer תא אפיתל; מוקדם התארכות מאוחר הפיכה העובר בצורת השעועית לתוך זחלים דמויים תולעת. בעקבות morphogenesis, צוהר עובר וזחלי L1 להתחיל להאכיל באמצעות חיידקים זמינים בסביבה המיידית שלהם. ההתארכות עוברית היא אפוא שלב מאוחר של ההתפתחות העוברית. הוא מורכב ההארכה העובר לאורך ציריו אורך הפחתה של הקוטר הרוחבי שלה. זה כרוך שינוי דרמטי של צורת התאים hypodermal. ההתארכות מחולקת צעיר ושלב מאוחר. השלב מוקדם מתחיל בשלב פסיק ומסתיים כאשר השרירים-קיר הגוף מתחיל לחלות בשלב 1.75 של פי wהכשרת הישוב מסוג (WT) עוברים - המתאים עוברים כי הם 1.75 פי אורך לעומת עוברים שאינם מוארך. תהליכים המתרחשים morphogenic בשלב זה מונעים בעיקר על ידי התכווצות של חבילות אקטין פילמנטיות (FBS) ממוקם בקוטב הפסגה של תאי hypodermal שמניעים התארכות שלהם לאורך ציר אנטרו-האחורי של העובר 2. התכווצות של FBS היא שליטה על ידי זרחון של שרשרות אור שרירן ידי שלוש קינאזות LET-502 / ROCK, MRCK-1 ו- PAK-1 5. השלב המאוחר של ההתארכות, מתחיל כאשר שרירי גוף לקיר להיות פונקציונליים ולהתחיל קבלנות. היא כרוכה איתות mechanotransduction מהשרירים-קיר הגוף אל hypodermal הגב ועל הגחון תאים ומסתיימת כאשר חיות לבקוע 3. פגמי התארכות מאופיינים לרוב אחוז בעלי החיים מתים כמו עוברי (הקטלני עוברי; Emb) ואלה התפתחותם מעצר כזחלי L1 (פנוטיפ מעצר זחל; Lvא) ולהיות קצרים משמעותי מאשר WT. זיהוי של השלב של מעצר התפתחותי דורש תצפית מיקרוסקופית של עוברים מתים ועוצר זחלי 3-6. היא הוצגה לאחרונה כי מספר גנים, כגון רגולטור Cdc42 / Rac ו Pix-1 המפעיל ו פאק-1, לשלוט בתהליכי morphogenic במהלך שני מוקדמת ומאוחר התארכות 3,7. הראנו גם לאחרונה שתהליכים morphogenic שונים לאורך ציר אנטרו-האחורי של העוברים במהלך התארכות מוקדם 3 7. ממצאים אלה הניעו את הפיתוח של מדדי רומן מיקוד בשלבי התארכות מוקדם או מאוחר במיוחד ומדדים אחרים, המאפשרים אפיון של המורפולוגיה של עובר לאורך ציר אנטרו-האחורי שלהם במהלך ההתארכות מוקדם. שיטות חדשניות אלה מורכבות במדידת האורך של עובר בתחילה ובסוף של התארכות מוקדם וכן את הרוחב שלהם הןמודעות וזנבות. 7 שני פרוטוקולים פותחו גם למדוד את אורך הזחלים שבקעו זה עתה, מסונכרן ב L1 בשלב 7. קליפות הביצים של עובר להגן עליהם מפני טיפול תת-כלורי אלקליין בעוד זחלים, מבוגרים חיידקים נוכחיים בתקשורת התרבות, נמוגים ידי הטיפול. טיפול זה משמש אז כדי לטהר עובר מתוך אוכלוסייה הלא מסונכרן המכילה מרבית מבוגרים הניזונים גם 8. הגבלת מזון משמשת לסנכרן זחלים שבקעו זה עתה. מדידת אורך הזחלים אלה לאחר מכן נעשה שימוש כדי לזהות פגמי התארכות. מדידה זו עדיפה על פני מדידת זחלים נעצרו על צלחות התרבית כי זחלים בוקעים מן הלא-לגמרי עוברים מוארכים יכולים להתאושש כדי &quot;אורך נורמלי&quot; כאשר האכלה אך ישמרו בגודל המוקטן שלהם כאשר נעצרו בהיעדר מזון. כאן, אנו מציגים פרוטוקולים מפורטים המאפשרים מדידה של length של מתארך עובר כמו גם את רוחב הראש שלהם וזנב באמצעות זמן לשגות דסק&quot;ש מיקרוסקופיה וניתוח תמונה (פרוטוקול 1). כמו כן אנו מספקים פרוטוקולים מפורטים כדי למדוד את אורך זחלים מסונכרנים באמצעות ניתוח תמונה (פרוטוקול 2) ושפל-Cytometry (פרוטוקול 3).

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			<td height="21" style="height:21px;width:216px;">glass slides</td>
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</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.

הראש, זנב והראש / יחס רוחב זנב הם מדדים חזקים. הפרוטוקולים המתואר כאן שימ?...

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

מדדי רומן לאפיון התארכות עוברית של נמטודות<em&gt; Elegans Caenorhabditis</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. ...

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Neuroscience

Novel Metrics to Characterize Embryonic Elongation of the Nematode Caenorhabditis elegans

6.0K Views.  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. 

Video: 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.

שיטות הקלטה בעל מספר יחידות לאפיין את הפעילות עצבית בארבה (<em&gt; Schistocerca אמריקנה</em&gt;) מעגלי ריח

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

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.

<em&gt; Elegans Caenorhabditis</em&gt; מערכת מודל לחקר Amylopathy

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.

שיטות לחקר מנגנוני פעולה של אנטי פסיכוטיות סמים ב<em&gt; Elegans Caenorhabditis</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.

Assay למדידת ההשפעות של אתנול במהירות התנועה של<em&gt; Elegans Caenorhabditis</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.

Microchambers agarose לסידן הדמיה לטווח ארוך של<em&gt; Elegans Caenorhabditis</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).

הדינמי כמוני בדיקה חושית לאפיון עיבוד כאב המרכזי

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.

א<em&gt; Caenorhabditis elegans</em&gt; מצב תזונתיים מבוססי Assay Assersion

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;

סידן רציומטרי הדמיה של נוירונים בודדים ב מתנהג Caenorhabditis Elegans

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

Combining Optogenetics with Artificial microRNAs to Characterize the Effects of Gene Knockdown on Presynaptic Function within Intact Neuronal Circuits

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

שילוב Optogenetics עם מיקרו Rna מלאכותי כדי לאפיין תופעות נוקאאוט גנטי על פונקציה Presynaptic בתוך מעגלים עצביים ללא פגע

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a ...

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.

הדמיה של קוצים דנדריטיים בקאנורהבדיטיס אלגנס

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