We thank Life Sciences and the Faculty of Arts and Science at Queen's University for funding this work. As well, we thank the Chin-Sang laboratory for providing the necessary reagents, equipment and technical support. We also thank QGEM 2011, especially Tony He for his contribution to the discussion.

1. Preparing/washing the Worms
<ol>
 <li>Synchronize worms to young adult11. </li>
 <li>Pipette 2 ml of the S Basal onto a 5 cm Chemotaxis plate of staged worms that have just cleared the lawn of OP50 E. coli. Tilt the plate as needed to ensure the worms are washed from the plate surface into the buffer. </li>
 <li>Pipette 1 ml of the worm-S Basal solution into a microcentrifuge tube. </li>
 <li>Centrifuge for 10 sec using a PicoFuge at 6,600 rpm.</li>
 <li>Aspirate the S Basal, leaving the pellet of worms undisturbed. </li>
 <li>Add 1 ml S Basal solution to the microcentrifuge tube, invert a few times to wash the worms. </li>
 <li>Repeat steps 1.4 to 1.6 another three times.</li>
 <li>Centrifuge for 10 sec a fifth time, this time aspirating the supernatant to a final volume of ca. 100 &mu;l.
 </li>
 <li>Pipette 2 &mu;l of the worms onto an NGM plate to ensure there are between 50 and 250 worms in each 2 &mu;l sample. Adjust the concentration of worms in the S Basal as needed by resuspending the worms in a smaller or larger volume of S Basal. 
 </li>
 <li>Use the worms in the assay immediately after washing the worms for up to 1 hr. </li>
</ol>
2. Preparing the Test Plates
<ol>
 <li>Marking the underside of a 5 cm plate, divide the dish into 4 equal quadrants. Chemotaxis agar or NGM may be used. A minimum of 3 trials are required per genetic strain being tested.
 </li>
 <li>Mark a circle of radius 0.5 cm around the origin (Figure 1).</li>
 <li>Mark a point in each quadrant with either a "T" for "Test" or a "C" for "Control," ensuring that the sites are equidistant from the origin and each other. The points must be at least 2 cm away from the origin. Mark the top left and bottom right quadrants as test quadrants and the top right and bottom left quadrants as controls (Figure 1). </li>
</ol>
3. Running the Assay
<ol>
 <li>Mix the test solution by combining equal volumes of the test compound and 0.5 M azide (an anesthetic used to arrest the worms upon reaching a quadrant). Mix the control solution by combining the solvent used to dilute the test compound with 0.5 M azide. </li>
 <li>Pipette 2 &mu;l of the worm solution (prepared in 1.8) from the pellet onto the origin (where the two lines intersect). </li>
 <li>Immediately after, pipette 2 &mu;l of the test solution onto the two "T" sites. Likewise, pipette the same amount of the control solution onto the two "C" sites. </li>
 <li>Once the worm and odorant drops have been absorbed in the agar, replace the lids and invert the plates. </li>
 <li>After 60 min, place the worms in a 4 &deg;C incubator. Only remove a plate from the incubator when it can be counted. Leaving worms at room temperature may allow them to mobilize again. </li>
 <li> Record the number of worms in each quadrant that completely crossed the inner circle. </li>
 <li> Repeat steps 3.2 to 3.6 using the control solution in both the test and control quadrants. This will serve as the control plate. Three such plates should be made and run to serve as an appropriate control. </li>
</ol>
4. Interpreting the Scores/Determining the Chemotaxis Index
<ol>
 <li>Calculate the chemotaxis index using Equation 1. This will yield a chemotactic index between -1.0 and +1.0.</li>
 </ol>
(1)
Chemotaxis Index = (# Worms in Both Test Quadrants - # Worms in Both Control Quadrants) / (Total # of Scored Worms) 
A +1.0 score indicates maximal attraction towards the target and represents 100% of the worms arriving in the quadrants containing the chemical target. An index of -1.0 is evidence of maximal repulsion. Similar assay methods have already been employed 3 (Table 1).
<ol start="2">
 <li>Report the mean Chemotaxis Index (CI) and standard error of mean (SEM). Perform a Student's T-test comparing the data from the test and control plates. </li>
 </ol>

<ol>
	<li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74, 515-527 (1993).</li><li>Bargmann, C. I., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensory+neurons+with+overlapping+functions+direct+chemotaxis+to+multiple+chemicals+in+C.+elegans.">Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C. elegans.</a> Neuron. 7, 729-742 (1991).</li><li>Lee, J., Jee, C., McIntire, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+preference+in+C.+elegans.">Ethanol preference in C. elegans.</a> Genes Brain Behav. 8, 578-585 (2009).</li><li>Swierczek, N. A., Giles, A. C., Rankin, C. H., Kerr, R. A. <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+behavioral+analysis+in+C.+elegans.">High-throughput behavioral analysis in C. elegans.</a> Nat. Methods. 8, 592-598 (2011).</li><li>Ward, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotaxis+by+the+nematode+Caenorhabditis+elegans:+identification+of+attractants+and+analysis+of+the+response+by+use+of+mutants.">Chemotaxis by the nematode Caenorhabditis elegans: identification of attractants and analysis of the response by use of mutants.</a> Proc. Natl. Acad. Sci. U.S.A. 70, 817-821 (1973).</li><li>Colbert, H. A., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-specific+adaptation+pathways+generate+olfactory+plasticity+in+C.+elegans.">Odorant-specific adaptation pathways generate olfactory plasticity in C. elegans.</a> Neuron. 14, 803-812 (1995).</li><li>Kauffman, A., Parsons, L., Stein, G., Wills, A., Kaletsky, R., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+Positive+Butanone+Learning,+Short-term,+and+Long-term+Associative+Memory+Assays.">C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays.</a> J. Vis. Exp. (49), e2490 (2011).</li><li>Troemel, E. R., Kimmel, B. E., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+chemotaxis+responses:+sensory+neurons+define+olfactory+preferences+in+C.+elegans.">Reprogramming chemotaxis responses: sensory neurons define olfactory preferences in C. elegans.</a> Cell. 91, 161-169 (1997).</li><li>Pierce-Shimomura, J. T., Morse, T. M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+role+of+pirouettes+in+Caenorhabditis+elegans+chemotaxis.">The fundamental role of pirouettes in Caenorhabditis elegans chemotaxis.</a> J Neurosci. 19, 9557-9569 (1999).</li><li>Frokjaer-Jensen, C., Ailion, M., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ammonium-acetate+is+sensed+by+gustatory+and+olfactory+neurons+in+Caenorhabditis+elegans.">Ammonium-acetate is sensed by gustatory and olfactory neurons in Caenorhabditis elegans.</a> PLoS One. 3, e2467 (2008).</li><li>Porta-de-la-Riva, M., Fontrodona, L., Villanueva, A., Cer&#243;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Caenorhabditis+elegans+Methods:+Synchronization+and+Observation.">Basic Caenorhabditis elegans Methods: Synchronization and Observation.</a> J. Vis. Exp. (64), e4019 (2012).</li><li>Sengupta, P., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+fate+specification+and+differentiation+in+the+nervous+system+of+Caenorhabditis+elegans.">Cell fate specification and differentiation in the nervous system of Caenorhabditis elegans.</a> Dev. Genet. 18, 73-80 (1996).</li><li>Hart, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior.">Behavior.</a> WormBook. , (1895).</li></ol>

We have nothing to disclose.

Chemotaxis, although controlled by a complex set of neuronal and cellular mechanisms, can be easily and objectively quantified using chemotaxis assays. To obtain the best results from the assays, certain critical steps must be taken. Firstly, staging the worms is essential in yielding consistent experimental results. Worms at different life stages behave differently 13; so mixed stage worms may skew experimental results. Secondly, ensuring all E. coli is washed off the worms is crucial, as residua...

Ward first developed the chemotaxis assay in 1973 5, and since then it has had far reaching applications. Neurobiology is one field that has benefitted from using a variety of chemotaxis assays. Olfactory adaptation, a simple form of learning and memory, has been demonstrated in C. elegans using chemotaxis assays 6. They have also been used to show that C. elegans can develop ethanol tolerance-a result that not only demonstrates the behavioral plasticity of the worms, but that also shows that the worms can be very useful in the study of alcohol dependence in humans 3. Assays have even been developed to demonstrate the ability of C. elegans to store short and long term memory by showing that associations are made by the worms between chemoattractants and food (OP50) 7. Additionally, given the extensive information currently available regarding the C. elegans genome, the chemotaxis behavior of C. elegans has been altered numerous times by inducing mutations 1,8. This allows for many exciting engineering possibilities, such as the development of C. elegans as a bioremediation tool. Thus, since the initial development of the chemotaxis assay in 1973, it has been frequently altered and used to elucidate mysteries in a variety of disciplines. 
Certain assays aimed to discover the specific route taken by the worms toward a target. The prototypical assay of this kind was developed by Ward 5. Three worms were placed on melted agar for 15 min. Their movements were traced by the imprint they left as they travelled from the periphery of the plate up a gradient to an attractant at the center of the plate. All worms on the plate were arrested using chloroform at the end of each trial. One descendant of this method placed a single worm in the middle of the plate with the attractant and the control at equal and opposite distances from the origin 2.
Pierce-Shimomura et al. developed an assay to observe the exact nature of the movement involved in chemotaxis9. Individual worms were placed in 9 cm Petri dishes either containing a uniform concentration of the attractant or a radial shaped gradient, culminating in the source of the attractant. A computer software program that recognized the worm was used to record the observed behavior. A video camera attached to a microscope worked in conjunction with the stage to adjust the Petri dish automatically as the assay ran to ensure the worm remained in the field of view. From this, more detailed information was discovered regarding the cause of pirouettes displayed by C. elegans.
Other assays, more similar to the one described here, tested the response of a large population of worms to test compounds. Two-quadrant chemotaxis assays have been used to explore the roles that various neurons, receptors, and signal transduction molecules played when C. elegans was exposed to various compounds 1. Between 20-50 washed worms were placed near the center of the plate with an attractant and a control at polar ends along with the anesthetic, sodium azide (NaN3). After 60 min, a chemotactic index with values from -1.0 to +1.0 was generated based on the difference between how many worms were affixed to the attractant or the control. A similar chemotactic index was used in the assay reported in this article, although the earlier assay failed to strictly evaluate non-motile worms. This assay was then further applied to testing the effects of neuronal ablation on chemotaxis. 
Another variation of the aforementioned assay was performed where 100-200 worms were placed at the center of a plate containing four quadrants 3. Adjacent quadrants either contained the test or control substance. As in previous assays, the worms were immobilized by the action of sodium azide before being scored. A similar method is described here as a way of evaluating the response of C. elegans to various compounds. However, the method below has the added benefit of only evaluating worms that have passed a threshold distance separating mobile from immobile worms.
Other assays have incorporated similar guidelines for ignoring immobile worms. Fr&oslash;kj&aelig;r-Jensen et al. developed a versatile assay which can be used to test both volatile and water soluble compounds 10. A Petri dish was divided into four quadrants. The top and bottom quadrants did not contain solvents. The left quadrant contained water, and the right contained the attractant. When testing volatile odorants, the analyte was placed on the lid of the dish, over the proper quadrant, whereas water soluble compounds were placed directly on the agar.
The methods currently in existence for evaluating the chemotactic response of C. elegans are constantly being refined to optimize their ease of use, efficiency, and accuracy. So, while the assay described here has the capability of assessing the greatest number of worms (maximum throughput: 250 worms/hour per plate, slightly greater than the throughput demonstrated by Lee et al. 3); the real strength of this method is the succinct culmination of many of the attributes of earlier assays (Table 1).

<table><tbody><tr><td>S Basal (- cholesterol) [5.8 g NaCl; 1 M K phosphate buffer, pH 6.0; dH&lt;sub&gt;2&lt;/sub&gt;O to 1 liter.] Autoclave</td><td></td><td></td><td></td></tr><tr><td>PicoFuge</td><td>Stratagene</td><td>400552</td><td></td></tr><tr><td>Microscopes</td><td>Leica</td><td></td><td></td></tr><tr><td>Dissecting</td><td>Leica</td><td></td><td></td></tr><tr><td>P1000 Pipette</td><td>Gilson</td><td></td><td></td></tr><tr><td>P10 Pipette</td><td>Gilson</td><td></td><td></td></tr><tr><td>0.5 M Sodium Azide</td><td></td><td></td><td></td></tr><tr><td>Chemotaxis Agar [1.6% BBL-agar (Benton-Dickinson) or 2% Difco-agar. Autoclave. Add 5 mM potassium phosphate, pH 6.0; 1 mM CaCl&lt;sub&gt;2&lt;/sub&gt;, 1 mM MgSO&lt;sub&gt;4&lt;/sub&gt;]</td><td></td><td></td><td></td></tr><tr><td>Ethanol/Distilled Water</td><td></td><td></td><td></td></tr><tr><td>Test Compounds (eg. 0.5% diacetyl)</td><td></td><td></td><td></td></tr><tr><td>Agar</td><td>Bio-Rad</td><td>166-0600</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Amresco</td><td>CA97062-046</td><td></td></tr><tr><td>MOPS</td><td>VWR</td><td>CA12001-120</td><td></td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;OH</td><td>BDH</td><td>CABDH8641-2</td><td></td></tr><tr><td>0.25% Tween 20</td><td>Bio-Rad</td><td>170-6531</td><td></td></tr></tbody></table>

c. elegans chemotaxis assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior.
 The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress 1-10. The method described here aims to address these issues by modifying the assay developed by Bargmann et al.1. A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

Comparing wild-type (N2) C. elegans to the odr-10(ky10) mutant.
We used diacetyl, a known C. elegans chemoattractant, to compare wildtype worms to that of a mutant that lacks the receptor for diacetyl 1,12. For wildtype (N2) worms the chemotactic index was 0.100±0.066 to ethanol, and 0.839±0.031 to 0.5% diacetyl. As expected, diacetyl elicits a significant chemoattractive response from wild-type worms (P&lt;0.003). In contrast the odr-10(ky10)<...

Watch this Scientific Journal Video about C. elegans Chemotaxis Assay at JoVE.com

C. elegans Chemotaxis Assay

A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

C. elegans Chemotaxis Assay

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates.  Caenorhabditis elegans has impressive chemotaxis ...

Research

JoVE Journal

Biology

45.6K Views.  Queen's University. A method of quantitatively evaluating the chemotactic response of Caenorhabditis elegans is described. A chemotactic index (CI) was employed as a way to precisely evaluate the response of worms to certain targets, and serve as a platform of comparison between strains and compounds of interest.

Video: C. elegans Chemotaxis Assay

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.

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

Neuroscience

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and pathways as well as its ease of cultivation. Several C. elegans disease models have been reported, including neurodegenerative disorders such as Parkinson's disease (PD), which involves the degeneration of dopaminergic (DA) neurons 1. Both transgenes and neurotoxic chemicals have been used to induce DA neurodegeneration and consequent movement defects in worms, allowing for investigations into the basis of neurodegeneration and screens for neuroprotective genes and compounds 2,3.
Screens in lower eukaryotes like C. elegans provide an efficient and economical means to identify compounds and genes affecting neuronal signaling. Conventional screens are typically performed manually and scored by visual inspection; consequently, they are time-consuming and prone to human errors. Additionally, most focus on cellular level analysis while ignoring locomotion, which is an especially important parameter for movement disorders.
We have developed a novel microfluidic screening system (Figure 1) that controls and quantifies C. elegans' locomotion using electric field stimuli inside microchannels. We have shown that a Direct Current (DC) field can robustly induce on-demand locomotion towards the cathode ("electrotaxis") 4. Reversing the field's polarity causes the worm to quickly reverse its direction as well. We have also shown that defects in dopaminergic and other sensory neurons alter the swimming response 5. Therefore, abnormalities in neuronal signaling can be determined using locomotion as a read-out. The movement response can be accurately quantified using a range of parameters such as swimming speed, body bending frequency and reversal time.
Our work has revealed that the electrotactic response varies with age. Specifically, young adults respond to a lower range of electric fields and move faster compared to larvae 4. These findings led us to design a new microfluidic device to passively sort worms by age and phenotype 6.
We have also tested the response of worms to pulsed DC and Alternating Current (AC) electric fields. Pulsed DC fields of various duty cycles effectively generated electrotaxis in both C. elegans and its cousin C. briggsae 7. In another experiment, symmetrical AC fields with frequencies ranging from 1 Hz to 3 KHz immobilized worms inside the channel 8.
Implementation of the electric field in a microfluidic environment enables rapid and automated execution of the electrotaxis assay. This approach promises to facilitate high-throughput genetic and chemical screens for factors affecting neuronal function and viability.

Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of Caenorhabditis elegans' Locomotion

A semi-automated micro-electro-fluidic method to induce on-demand locomotion in Caenorhabditis elegans is described. This method is based on the neurophysiologic phenomenon of worms responding to mild electric fields (&#x201C;electrotaxis&#x201D;) inside microfluidic channels. Microfluidic electrotaxis serves as a rapid, sensitive, low-cost, and scalable technique to screen for factors affecting neuronal health.

The nematode Caenorhabditis elegans is a versatile model organism for biomedical research because of its conservation of disease-related genes and ...

Bioengineering

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Behavior

Extraction and Quantification of Volatile Sex Pheromone Responses in C. elegans

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

Author Spotlight: Examining Volatile Sex Pheromone Influence on Male C. elegans Behavior

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

Measurement of Cellular Chemotaxis with ECIS/Taxis

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to infection. A common method to measure chemotaxis is the Boyden chamber assay, in which cells and chemoattractant are separated by a porous membrane. As cells migrate through the membrane toward the chemoattractant, they adhere to the underside of the membrane, or fall into the underlying media, and are subsequently stained and visually counted 1. In this method, cells are exposed to a steep and transient chemoattractant gradient, which is thought to be a poor representation of gradients found in tissues 2.
Another assay system, the under-agarose chemotaxis assay, 3, 4 measures cell movement across a solid substrate in a thin aqueous film that forms under the agarose layer. The gradient that develops in the agarose is shallow and is thought to be an appropriate representation of naturally occurring gradients. Chemotaxis can be evaluated by microscopic imaging of the distance traveled. Both the Boyden chamber assay and the under-agarose assay are usually configured as endpoint assays.
The automated ECIS/Taxis system combines the under-agarose approach with Electric Cell-substrate Impedance Sensing (ECIS) 5, 6. In this assay, target electrodes are located in each of 8 chambers. A large counter-electrode runs through each of the 8 chambers (Figure 2). Each chamber is filled with agarose and two small wells are the cut in the agarose on either side of the target electrode. One well is filled with the test cell population, while the other holds the sources of diffusing chemoattractant (Figure 3). Current passed through the system can be used to determine the change in resistance that occurs as cells pass over the target electrode. Cells on the target electrode increase the resistance of the system 6. In addition, rapid fluctuations in the resistance represent changes in the interactions of cells with the electrode surface and are indicative of ongoing cellular shape changes. The ECIS/Taxis system can measure movement of the cell population in real-time over extended periods of time, but is also sensitive enough to detect the arrival of a single cell at the target electrode. 
Dictyostelium discoidium is known to migrate in the presence of a folate gradient 7, 8 and its chemotactic response can be accurately measured by ECIS/Taxis 9. Leukocyte chemotaxis, in response to SDF1&alpha; and to chemotaxis antagonists has also been measured with ECIS/Taxis 10, 11. An example of the leukocyte response to SDF1&alpha; is shown in Figure 1.

The ECIS/Taxis system is an automated, real-time assay that measures cellular chemotaxis. In this assay, cells move beneath a layer of agarose to arrive at a target electrode. Cellular movement is measured by the onset of resistance to AC current 0.

Cellular movement in response to external stimuli is fundamental to many cellular processes including wound healing, inflammation and the response to ...

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an attractive model organism to study learning and memory at molecular and cellular levels because of the simplicity of its nervous system, whose chemical and electrical wiring diagrams were completely reconstructed from serial electron micrographs of thin sections. Here, we describe detailed protocols for the conditioning of C. elegans by massed and spaced training for the formation of short-term memory (STM) and long-term memory (LTM), respectively. By pairing 1-propanol and hydrochloric acid as conditioned and unconditioned stimuli, respectively, C. elegans was successfully trained to form aversive associative STM and LTM. While na&#239;ve animals were attracted to 1-propanol, the trained animals were no longer or very weakly attracted to 1-propanol. Like in other organisms such as Aplysia and Drosophila, "learning and memory genes" play essential roles in memory formation. Particularly, NMDA-type glutamate receptors, expressed in only six pairs of interneurons in C. elegans, are required for the formation of both STM and LTM, possibly as a coincidence factor. Therefore, the memory trace may reside among the interneurons.

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

We have previously developed protocols for Caenorhabditis elegans to form short- and long-term associative memories by massed and spaced training, respectively. Here, detailed protocols are described for the conditioning of C. elegans by pairing 1-propanol and hydrochloric acid as conditioned and unconditioned stimuli, respectively, to form aversive associative memory.

The nematode Caenorhabditis elegans is an attractive model organism to study learning and memory at molecular and cellular levels because of the ...

Chemotaxis is a process in which cells or organisms move in response to a chemical stimulus. In nature, chemotaxis is important for organisms to sense and move toward food sources and move away from stimuli that may be toxic or harmful. Chemotaxis is also important at the cellular level. For example, chemotaxis is required for the movement of sperm toward an egg prior to fertilization. In the lab, chemotaxis is frequently examined in the nematode, C. elegans, which is known to migrate towards food sources in soil, but away from toxins such as heavy metals, substances with a low pH, and detergents. This video demonstrates how to perform a chemotaxis assay, which includes preparing the chemotaxis plates and the worms, running the assay, and analyzing the data. Then, we discuss examples of how chemotaxis assays can be used in C. elegans as a tool to understand learning and memory, olfactory adaptation, and neurological disease such as Alzheimer&quot;s disease. Chemotaxis experiments in C. elegans have near-limitless possibilities for learning more about the cellular and genetic mechanisms of many biological processes, and may lead to a greater understanding of human biology, development, and disease.

Chemotaxis Assay in Caenorhabditis elegans

Chemotaxis is a process in which cells or organisms move in response to a chemical stimulus. In nature, chemotaxis is important for organisms to sense and ...

Education

JoVE Science Education

Basic Biology

Biology I: yeast, Drosophila and C. elegans

C. elegans Survival Assay: A Short-Term Toxicity Test

Source: Possik, E. and Pause, A. <a href="https://www.jove.com/video/2496/measuring-caenorhabditis-elegans-life-span-96-well-microtiter">Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.</a>&#160;J. Vis. Exp. (2015).
This video introduces a short-term survival assay that tests a treatment for acute toxicity or tests a genetic strain for resistance to a toxic condition.&#160; The example protocol shows an example survival assay in which worms are tested for their ability to&#160; resist oxidative stress induced by paraquat (PQ).

Source: Possik, E. and Pause, A. Measuring Oxidative Stress Resistance of&#160;Caenorhabditis elegans&#160;in 96-well Microtiter Plates.&#160;J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Caenorhabditis elegans (worm)

Anatomy and Physiology

C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms

Source: Campbell, J. C., et al. <a href="https://www.jove.com/video/55939/a-caenorhabditis-elegans-nutritional-status-based-copper-aversion">A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay.</a> J. Vis. Exp. (2017).
This video introduces chemotaxis in the nematode C. elegans and shows a sample protocol testing chemotactic aversion to copper sulfate.

Source: Campbell, J. C., et al. A Caenorhabditis elegans Nutritional-Status Based Copper Aversion Assay. J. Vis. Exp. (2017).
This video introduces ...

Electrotaxis Assay: A Method to Observe Locomotion in C. elegans

Source: Tong, J,&#160;et al. <a href="https://www.jove.com/video/50226/microfluidic-based-electrotaxis-for-on-demand-quantitative-analysis">Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of&#160;Caenorhabditis elegans&#39; Locomotion.</a>&#160;J. Vis. Exp.&#160;(2013).
Researchers can trigger on-demand movement in C. elegans by applying an electric field that worms sense and respond to.&#160; This video introduces electrotactic behavior and shows a sample protocol that is done in a microfluidic device.

Source: Tong, J,&#160;et al. Microfluidic-based Electrotaxis for On-demand Quantitative Analysis of&#160;Caenorhabditis elegans&#39; Locomotion.&#160;J. ...

C. elegans Chemotaxis Assay

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Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

C. elegans Chemotaxis Assay

C. elegans Survival Assay: A Short-Term Toxicity Test

C. elegans Chemotaxis Assay: A Method to Test Chemosensation in Worms

Electrotaxis Assay: A Method to Observe Locomotion in C. elegans