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 border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>S Basal (- cholesterol) [5.8 g NaCl; 1 M K phosphate buffer, pH 6.0; dH2O to 1 liter.] Autoclave</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PicoFuge</td>
 <td>Stratagene</td>
 <td>400552</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microscopes</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dissecting</td>
 <td>Leica</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>P1000 Pipette</td>
 <td>Gilson</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>P10 Pipette</td>
 <td>Gilson</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.5 M Sodium Azide</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</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 CaCl2, 1 mM MgSO4]</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ethanol/Distilled Water</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Test Compounds (eg. 0.5% diacetyl)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Agar</td>
 <td>Bio-Rad</td>
 <td>166-0600</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NH4Cl</td>
 <td>Amresco</td>
 <td>CA97062-046</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>MOPS</td>
 <td>VWR</td>
 <td>CA12001-120</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NH4OH</td>
 <td>BDH</td>
 <td>CABDH8641-2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.25% Tween 20</td>
 <td>Bio-Rad</td>
 <td>170-6531</td>
 <td>&nbsp;</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&plusmn;0.066 to ethanol, and 0.839&plusmn;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 o...

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 ...

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45.4K 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

Generation of Stable Transgenic C. elegans Using Microinjection

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1. The plasmid DNA rearranges to form extrachromosomal concatamers that are stably inherited, though not with the same efficiency as actual chromosomes 2. A gene of interest is co-injected with an obvious phenotypic marker, such as rol-6 or GFP, to allow selection of transgenic animals under a dissecting microscope. The exogenous gene may be expressed from its native promoter for cellular localization studies. Alternatively, the transgene can be driven by a different tissue-specific promoter to assess the role of the gene product in that particular cell or tissue. This technique efficiently drives gene expression in all tissues of C. elegans except for the germline or early embryo 3. Creation of transgenic animals is widely utilized for a range of experimental paradigms. This video demonstrates the microinjection procedure to generate transgenic worms. Furthermore, selection and maintenance of stable transgenic C. elegans lines is described.

This video demonstrates the technique of microinjection into the gonad of C. elegans to create transgenic animals.

Transgenic Caenorhabditis elegans can be readily created via microinjection of a DNA plasmid solution into the gonad 1.  The plasmid DNA rearranges to ...

Application of a C. elegans Dopamine Neuron Degeneration Assay for the Validation of Potential Parkinson's Disease Genes

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations at risk. In the process of attempting to identify novel genetic factors associated with PD, scientists have generated many lists of candidate genes, polymorphisms, and proteins that represent important advances, but these leads remain mechanistically undefined. Our work is aimed toward significantly narrowing such lists by exploiting the advantages of a simple animal model system. While humans have billions of neurons, the microscopic roundworm Caenorhabditis elegans has precisely 302, of which only eight produce dopamine (DA) in hemaphrodites. Expression of a human gene encoding the PD-associated protein, alpha-synuclein, in C. elegans DA neurons results in dosage and age-dependent neurodegeneration.
Worms expressing human alpha-synuclein in DA neurons are isogenic and express both GFP and human alpha-synuclein under the DA transporter promoter (Pdat-1). The presence of GFP serves as a readily visualized marker for following DA neurodegeneration in these animals. We initially demonstrated that alpha-synuclein-induced DA neurodegeneration could be rescued in these animals by torsinA, a protein with molecular chaperone activity 1. Further, candidate PD-related genes identified in our lab via large-scale RNAi screening efforts using an alpha-synuclein misfolding assay were then over-expressed in C. elegans DA neurons. We determined that five of seven genes tested represented significant candidate modulators of PD as they rescued alpha-synuclein-induced DA neurodegeneration 2. Additionally, the Lindquist Lab (this issue of JoVE) has performed yeast screens whereby alpha-synuclein-dependent toxicity is used as a readout for genes that can enhance or suppress cytotoxicity. We subsequently examined the yeast candidate genes in our C. elegans alpha-synuclein-induced neurodegeneration assay and successfully validated many of these targets 3, 4.
Our methodology involves generation of a C. elegans DA neuron-specific expression vector using recombinational cloning of candidate gene cDNAs under control of the Pdat-1 promoter. These plasmids are then microinjected in wild-type (N2) worms, along with a selectable marker for successful transformation. Multiple stable transgenic lines producing the candidate protein in DA neurons are obtained and then independently crossed into the alpha-synuclein degenerative strain and assessed for neurodegeneration, at both the animal and individual neuron level, over the course of aging.

This video demonstrates how to use C. elegans to assess dopaminergic neuron neurodegeneration as a model for Parkinson's disease. Furthermore, genetic screens are used to identify factors that either enhance degeneration or are neuroprotective.

Improvements to the diagnosis and treatment of Parkinson's disease (PD) are dependent upon knowledge about susceptibility factors that render populations ...

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the anatomy and connectivity of its nervous system has been determined from electron micrographs and refined by pharmacological assays. In this video, we describe how two complementary neural stimulants, an acetylcholinesterase inhibitor, called aldicarb, and a gamma-aminobutyric acid (GABA) receptor antagonist, called pentylenetetrazole (PTZ), may be employed to specifically characterize signaling at C. elegans neuromuscular junctions (NMJs) and facilitate our understanding of antagonistic neural circuits.
Of 302 C. elegans neurons, nineteen GABAergic D-type motor neurons innervate body wall muscles (BWMs), while four GABAergic neurons, called RMEs, innervate head muscles. Conversely, thirty-nine motor neurons express the excitatory neurotransmitter, acetylcholine (ACh), and antagonize GABA transmission at BWMs to coordinate locomotion. The antagonistic nature of GABAergic and cholinergic motor neurons at body wall NMJs was initially determined by laser ablation and later buttressed by aldicarb exposure. Acute aldicarb exposure results in a time-course or dose-responsive paralysis in wild-type worms. Yet, loss of excitatory ACh transmission confers resistance to aldicarb, as less ACh accumulates at worm NMJs, leading to less stimulation of BWMs. Resistance to aldicarb may be observed with ACh-specific or general synaptic function mutants. Consistent with antagonistic GABA and ACh transmission, loss of GABA transmission, or a failure to negatively regulate ACh release, confers hypersensitivity to aldicarb. Although aldicarb exposure has led to the isolation of numerous worm homologs of neurotransmission genes, aldicarb exposure alone cannot efficiently determine prevailing roles for genes and pathways in specific C. elegans motor neurons. For this purpose, we have introduced a complementary experimental approach, which uses PTZ.
Neurotransmission mutants display clear phenotypes, distinct from aldicarb-induced paralysis, in response to PTZ. Wild-type worms, as well as mutants with specific inabilities to release or receive ACh, do not show apparent sensitivity to PTZ. However, GABA mutants, as well as general synaptic function mutants, display anterior convulsions in a time-course or dose-responsive manner. Mutants that cannot negatively regulate general neurotransmitter release and, thus, secrete excessive amounts of ACh onto BWMs, become paralyzed on PTZ. The PTZ-induced phenotypes of discrete mutant classes indicate that a complementary approach with aldicarb and PTZ exposure paradigms in C. elegans may accelerate our understanding of neurotransmission. Moreover, videos demonstrating how we perform pharmacological assays should establish consistent methods for C. elegans research.

This video demonstrates how to employ two neural stimulants, aldicarb and pentylenetetrazole (PTZ), in complementary ways to study synaptic function in the nematode, C. elegans. This complementary approach may also be used to shed light on evolutionarily conserved mechanisms for modulating neuronal synchrony and has implications for epilepsy and seizures.

The nematode, Caenorhabditis elegans, has become an expedient model for studying neurotransmission. C. elegans is unique among animal models, as the ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes. However, many compounds identified in screens using cell culture models are often found to be toxic or pharmacologically inactive in vivo1-2. Screening in whole animal models can help avoid these pitfalls and streamline the path to drug development.
C. elegans is a multicellular model organism well suited for HTS. It is small (&lt;1 mm) and can be economically cultured and dispensed in liquids. C. elegans is also one of the most experimentally tractable animal models permitting rapid and detailed identification of drug mode-of-action3.
We describe a protocol for culturing and dispensing fluorescent strains of C. elegans for high-throughput screening of chemical libraries or detection of environmental contaminants that alter the expression of a specific gene. Large numbers of developmentally synchronized worms are grown in liquid culture, harvested, washed, and suspended at a defined density. Worms are then added to black, flat-bottomed 384-well plates using a peristaltic liquid dispenser. Small molecules from a chemical library or test samples (e.g., water, food, or soil) can be added to wells with worms. In vivo, real-time fluorescence intensity is measured with a fluorescence microplate reader. This method can be adapted to any inducible gene in C. elegans for which a suitable reporter is available. Many inducible stress and developmental transcriptional pathways are well defined in C. elegans and GFP transgenic reporter strains already exist for many of them4. When combined with the appropriate transgenic reporters, our method can be used to screen for pathway modulators or to develop robust biosensor assays for environmental contaminants. 
We demonstrate our C. elegans culture and dispensing protocol with an HTS assay we developed to monitor the C. elegans cap &#x2018;n&#x2019; collar transcription factor SKN-1. SKN-1 and its mammalian homologue Nrf2 activate cytoprotective genes during oxidative and xenobiotic stress5-10. Nrf2 protects mammals from numerous age-related disorders such as cancer, neurodegeneration, and chronic inflammation and has become a major chemotherapeutic target11-13.Our assay is based on a GFP transgenic reporter for the SKN-1 target gene gst-414, which encodes a glutathione-s transferase6. The gst-4 reporter is also a biosensor for xenobiotic and oxidative chemicals that activate SKN-1 and can be used to detect low levels of contaminants such as acrylamide and methyl-mercury15-16.

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

A procedure for liquid-based culturing and dispensing of C. elegans strains expressing fluorescent reporter proteins is described that does not require expensive sorting equipment. This approach can be applied to numerous inducible C. elegans genes for drug discovery or biosensing of contaminants.

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

A Method for Culturing Embryonic C. elegans Cells

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require direct access to cells and isolation of specific cell types, could not be applied in C. elegans. This limitation was due to the fact that tissues are confined within a pressurized cuticle which is not easily digested by treatment with enzymes and/or detergents. Based on early pioneer work by Laird Bloom, Christensen and colleagues 1 developed a robust method for culturing C. elegans embryonic cells in large scale. Eggs are isolated from gravid adults by treatment with bleach/NaOH and subsequently treated with chitinase to remove the eggshells. Embryonic cells are then dissociated by manual pipetting and plated onto substrate-covered glass in serum-enriched media. Within 24 hr of isolation cells begin to differentiate by changing morphology and by expressing cell specific markers. C. elegans cells cultured using this method survive for up 2 weeks in vitro and have been used for electrophysiological, immunochemical, and imaging analyses as well as they have been sorted and used for microarray profiling.

A Method for Culturing Embryonic C. elegans Cells

We describe here a revised protocol for large-scale culture of embryonic C. elegans cells. Embryonic C. elegans cells cultured in vitro using this method, appear to differentiate and recapitulate the expression of genes in a cell specific manner. Techniques that require direct access to the cells or isolation of specific cell types from the other tissues can be applied on C. elegans cultured cells.

C. elegans is a powerful model system, in which genetic and molecular techniques are easily applicable. Until recently though, techniques that require ...

Antibody Staining in C. Elegans Using &quot;Freeze-Cracking&quot;

To stain C. elegans with antibodies, the relatively impermeable cuticle must be bypassed by chemical or mechanical methods. "Freeze-cracking" is one method used to physically pull the cuticle from nematodes by compressing nematodes between two adherent slides, freezing them, and pulling the slides apart. Freeze-cracking provides a simple and rapid way to gain access to the tissues without chemical treatment and can be used with a variety of fixatives. However, it leads to the loss of many of the specimens and the required compression mechanically distorts the sample. Practice is required to maximize recovery of samples with good morphology. Freeze-cracking can be optimized for specific fixation conditions, recovery of samples, or low non-specific staining, but not for all parameters at once. For antibodies that require very hard fixation conditions and tolerate the chemical treatments needed to chemically permeabilize the cuticle, treatment of intact nematodes in solution may be preferred. If the antibody requires a lighter fix or if the optimum fixation conditions are unknown, freeze-cracking provides a very useful way to rapidly assay the antibody and can yield specific subcellular and cellular localization information for the antigen of interest.

Antibody Staining in C. Elegans Using "Freeze-Cracking"

"Freeze-cracking," a method for exposing the inner tissues of the nematode C. elegans to antibodies for protein localization, is demonstrated.

To stain C.  elegans with antibodies, the relatively impermeable cuticle must be  bypassed by chemical or mechanical methods.  "Freeze-cracking" is one ...