Name: measuring caenorhabditis elegans sensitivity to the acetylcholine receptor agonist levamisole
Uploaded: 2022-06-07T14:00:00.000+00:00
Duration: 8 min 11 s
Description: Watch this Scientific Journal Video about Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole at JoVE.com

We thank Riya Dattani and Lauren Hogstrom, who collected the data in Figure 2B,C blind to genotype in the BISC413 Advanced Genetics Laboratory (Spring 2022) at the University of Delaware. Additional information about the BISC413 course-based undergraduate research experience (CURE), as well as access to the lab manual designed by J. Tanis, is available upon request. Nematode strains were provided by the Caenorhabditis Genetics Center, which is supported by the NIH-ORIP (P40 OD010440). This work was supported by a P20 GM103446 Delaware INBRE Mentored CURE Award (to J.E.T.).

1. Preparation of plates for the levamisole assay
<ol>
	<li>Prepare nematode growth medium (NGM) media by combining 3 g of NaCl, 2.5 g of peptone, and 17 g of agar with 1 L of deionized (DI) water in a flask with a stir bar.</li>
	<li>After autoclaving, put the media on a hot plate set to 70 &#176;C and stir at a moderate speed for 1 h.</li>
	<li>Add 1 mL of 5 mg/mL cholesterol dropwise to prevent precipitation, 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, and 25 mL of 1 M pH 6.0 potassium phosphate buffer&#160;to the media.</li>
	<li>Transfer 2 mL of NGM media into each well of a 24-well plate with a sterile serological pipette (Figure 1).</li>
	<li>Allow the plates to dry on the benchtop for 2 days before seeding with bacteria. For longer-term storage, place them at 4 &#176;C.</li>
	<li>Streak out OP50 E. coli on a lysogeny broth (LB) plate and grow overnight at 37 &#176;C.</li>
	<li>Pick a single OP50 colony into B-broth (10 g of tryptone and 5 g of NaCl in 1 L of DI H2O) and set the culture shaking at 37 &#176;C overnight.</li>
	<li>Using a sterile pipet, drop 30 &#181;L of OP50 suspension onto the agar in the middle of each well (Figure 1). 
	NOTE: Be sure not to damage the agar surface as this will lead to worm burrowing.</li>
	<li>Let the plates sit at room temperature for at least 2 days after seeding to allow a bacterial lawn to form. 
	NOTE: If the bacteria in the center wells is not dry after 2 days, leave the plates open in the hood for 20 min (Figure 1). The plates should be poured and seeded with bacteria 1-2 weeks before the assay.</li>
</ol>
2. Synchronizing&#160;&#160;C. elegans (day 1)
<ol>
	<li>Grow wild-type,&#160;unc-63(x26),&#160;lev-10(x17), and unc-49(e407) C. elegans to adulthood on 6 cm plates, preparing at least eight plates per strain. Confirm that there are many non-starved gravid adults on the plates. 
	NOTE: This is twice as many plates as needed; however, it is important to have backup plates in case the first batch of eggs is destroyed during bleaching.</li>
	<li>Make 10x M9 buffer by dissolving 59.6 g of Na2HPO4 (dibasic),&#160;29.9 g of KH2PO4&#160;(monobasic), 12.8 g of NaCl, and 2.5 g of MgSO4 in 750 mL of DI water with stirring. Once dissolved, bring the volume up to 1 L and filter sterilize. Dilute 1:10 and autoclave to make 1x M9 buffer. 
	NOTE: The 1x M9 buffer can be made weeks or months in advance.</li>
	<li>Prepare bleaching solution under the hood on the day of synchronization. Mix 10 mL of bleach (Table of Materials), 2.5 mL of 10 N NaOH, and 37.5 mL of DI water in a 50 mL conical tube. Wear goggles, gloves, and a lab coat whenever working with the bleaching solution.</li>
	<li>Using a plastic transfer pipet, wash gravid adult worms from at least four plates with 1x M9 buffer and transfer them into a 15 mL conical tube.</li>
	<li>Spin at 716 x g for 1 min at room temperature, and then remove the supernatant using a transfer pipet.</li>
	<li>Add 10 mL of the bleaching solution. Invert or gently shake the tube for ~4 min until most, but not all, of the worm carcasses have dissolved (Figure 1). 
	NOTE: Be careful not to over-bleach the worms as this will destroy the eggs. Certain mutations may increase the difficulty of egg isolation.</li>
	<li>Spin at 716 x g for 1 min.</li>
	<li>Pour off the bleach solution in one motion; as long as the tube is not shaken at this point, the eggs will stick to the side of the tube.</li>
	<li>Add 15 mL of 1x M9 buffer and invert. Spin at 716 x g for 1 min, and pour off M9 buffer in one smooth motion.</li>
	<li>Perform three washes in total with 1x M9 buffer, as described in step 2.9.</li>
	<li>After the final wash, add 10 mL of fresh 1x M9 and place on a rotator overnight at 15 &#176;C to isolate a synchronized population of starved first larval (L1) stage animals. 
	NOTE: Before placing on the rotator, check to make sure that there are eggs in the M9 buffer. If not, repeat the prep with backup plates and bleach for a shorter time.</li>
</ol>
3. Plating synchronized&#160;&#160;C. elegans (day 2)
<ol>
	<li>Print out a 24-well plate map and assign strains to randomized places. Each strain should be represented in the 24-well plate at least six times.</li>
	<li>Approximately 24 h after the bleach prep, spin down hatched starved L1 worms in&#160;M9 buffer at 716 x g for 1 min at room temperature.</li>
	<li>Remove ~9 mL of M9 buffer with a plastic transfer pipet, and then gently mix the starved first larval stage worms (L1s) in the remaining M9 buffer.</li>
	<li>Immediately pipet 3 &#181;L of the worms in M9 buffer onto a microscope slide and determine the number of L1s; the desired number is 20-30 L1s in 3 &#181;L. Spin down and remove M9 if the worm concentration is too low, and add M9 if the concentration is too high. 
	NOTE: Check the number of worms per 3 &#181;L at least 2x, inverting the tube each time.</li>
	<li>Pipet 3 &#181;L of L1s (20-30 worms total) into each well according to the pre-made plate map (step 3.1; Figure 1). 
	NOTE: Invert the tube with the L1s in M9 frequently to maintain an even distribution of worms as they will settle to the bottom. If this is not done, some wells will have too few worms, while others will have too many. Also, do not pierce the agar with the pipet tip as this will cause the animals to burrow.</li>
	<li>Let the worms grow to adulthood for 3 days at 20 &#176;C. 
	&#8203;NOTE: The use of different growth temperatures and certain mutations can impact maturation speed, so be sure to consider the C. elegans life cycle.</li>
</ol>
4. Performing the levamisole assay (day 5)
<ol>
	<li>Print a blank datasheet, which will be used to record the number of worms moving in each well every 5 min for 1 h. 
	NOTE: Students will be able to count the worms in at most 12 wells every 5 min. The top 12 wells are assayed in the first hour, with the bottom 12 wells assayed in the subsequent hour.</li>
	<li>Check the worms in the 24-well plates. Using a marker, make an &#34;X&#34; on the plate lid over any wells that have contamination, have starved, or have too many worms (&#62;40), which will make counting difficult.</li>
	<li>Make 0.4 mM levamisole solution by adding 200 &#181;L of 100 mM levamisole stock to 50 mL of 1x M9. Prepare 100 mM levamisole stock by dissolving 240.76 mg of levamisole hydrochloride in 10 mL of H2O and store it at &#8722;20 &#176;C. 
	NOTE: Levamisole must be diluted in M9; dilution in H2O accelerates paralysis. Students should wear gloves. Ingestion of high concentrations of levamisole is toxic.</li>
	<li>Start a timer and then, using a transfer pipet, add 1 mL of 0.4 mM levamisole to the first two wells, such that the animals are freely swimming. Continue to add levamisole to the adjacent wells, staggering the time according to the number of wells to be assayed. 
	NOTE: For example, if 10 wells are assayed, levamisole will be added in the following manner: wells 1 and 2 at time 0, wells 3 and 4 after 1 min, wells 5 and 6 after 2 min, wells 7 and 8 after 3 min, and wells 9 and 10 after 4 min.</li>
	<li>At 5 min, start manually counting only the number of moving worms in each well, beginning with the first well, and record that number on the datasheet (Figure 1). Counters can be used but are not required to accurately determine the number of moving worms. 
	NOTE: Students need to keep an eye on the timer as they will sometimes get behind during early time points before many worms paralyze. The number of time points can be adjusted, or fewer wells can be assayed at each time point if necessary.</li>
	<li>Continue to count the number of moving worms in each well every 5 min for 1 h. 
	NOTE: Immersion in M9 induces constant swimming movement over the course of this 1 h assay, which eliminates the need for the prodding of the worms to assess paralysis (Figure 2A). Exposure to 0.4 mM levamisole solution induces time-dependent paralysis as observed by cessation of swimming; worms that paralyze do not recover.</li>
	<li>Repeat steps 4.4.-4.6. for the rest of the wells in the plate.</li>
	<li>At the end of the assay, or when time permits, record the total number of worms in each well.</li>
</ol>
5. Data analysis
<ol>
	<li>Obtain the plate map (step 3.1.) and record which strain corresponds to each well. 
	NOTE: Due to the amount of data collected, the subsequent analysis and discussion of the data will take at least a couple of hours.</li>
	<li>Enter the data into a spreadsheet, starting with the total number of worms in each well and organizing by genotype.</li>
	<li>Combining data from the wells, determine the number of worms moving at each time point for every strain.</li>
	<li>Use these data to create a &#34;survival curve&#34; in the statistical software (Table of Materials) to visually display the time-dependent paralysis of the population (Figure 2B).</li>
	<li>Make a data table such that the left-hand column indicates time and subsequent columns contain the data for each strain. For each animal that is paralyzed within the first 5 min, create a row and enter a &#34;1&#34; for 5 min. Repeat this for each time point. For all animals that do not paralyze by the end of the assay, enter a &#34;0&#34; for 60 min.</li>
	<li>Perform pairwise comparisons using the log-rank (Mantel-Cox) test in the statistical software (Table of Materials) to determine if there is a statistically significant difference between two different strains.</li>
	<li>For additional/alternative analysis, instead of performing step 5.3. and step 5.4., determine the percentage of animals moving in each well at every time point. Plot the average with standard error at each time point for all strains assayed using a spreadsheet program or the statistical software (Figure 2C).</li>
</ol>

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The authors have no conflict of interest.

Altered response to levamisole can identify genes required for postsynaptic cholinergic signaling and muscle function. The protocol here describes a liquid levamisole assay used to quantitate time-dependent paralysis over a 1 h time period. Students make predictions about the effects of certain genetic mutations, carry out an experiment with large sample size, and then quantitate their results. This assay is a simple, efficient way to quantitate levamisole-induced paralysis without picking or prodding animals, making it suitable for undergraduate laboratories, as well as researchers studying neuromuscular transmission. Discussed here are some of the most common pitfalls, the limitations of the assay, and a variation to the protocol that enables its use with RNAi knockdown animals; also discussed here is how this assay can be embedded into a course-based undergraduate research experience.
Proper preparation of the 24-well plates is essential. First, bacterial lawns must be completely dry before spotting L1 animals in the wells. If not dried enough, the bacteria mixes with the levamisole solution during the assay, turning the liquid cloudy and preventing individuals from being able to count the worms. Second, when synchronizing worms through bleach prep, the animals must not be exposed to the bleach solution for too long as this will cause the protective coating on the eggs to disintegrate. Third, it is crucial to spot only 20-30 L1s per well, as too many worms in the wells make it extremely difficult to accurately count the number moving in the time allotted. Finally, it is important to maintain an aseptic technique during the preparation of the plates as contaminated wells cannot be assayed. 
There are a few limitations that must be considered when performing this assay. First, counting the number of moving worms in each well every 5 min may be overwhelming for individuals that lack prior lab experience, and this could lead to imprecise data collection. As for other assays used to determine drug sensitivity18,19, this experiment can be performed with fewer time points. Second, the plating of starved L1s isolated from a bleach prep is an easy method to obtain a synchronized population; however, adjustments must be made if the developmental timing differs between strains being assayed. It is possible to pick late fourth larval stage animals (L4s) into a 24-well plate for this assay; however, when preparing many plates, this is too labor-intensive. Alternatively, one should determine the length of time it takes for each strain to reach L4 and then place the L1s into the wells at different times to adjust for differences in maturation speed. Finally, while this assay can be used to identify new genes important for postsynaptic muscle function11, altered levamisole response can also be caused by mutation or RNAi knockdown of presynaptic genes12. If altered levamisole sensitivity is observed, additional experiments must be performed to determine the reason for this phenotype.
By making a few modifications to the 24-well plates, the described levamisole swim assay can also be performed on RNAi knockdown animals11. Gene knockdown through RNAi can be achieved by feeding C. elegans bacteria that express double-stranded RNA corresponding to the gene of interest20. For the preparation of RNAi plates, 1 mL of 1M IPTG and 1 mL of 25 mg/mL carbenicillin must be added per liter of media after removal from the autoclave. Bacterial cultures are grown by picking a single colony into 3 mL of LB broth plus 3 &#181;L of 25 mg/mLcarbenicillin, and shaking at 37 &#176;C overnight, and the plate map is made when the different bacteria are spotted in the wells. C. elegans are synchronized by bleach prep as described; however, the RNAi hypersensitive eri-1(mg366) strain should be used instead of the wild type to increase gene knockdown. RNAi knockdowns result in the same levamisole phenotypes observed for loss-of-function mutants11. 
The protocol presented here can be used in laboratory research, as a stand-alone experiment in the undergraduate lab, or in the opening weeks of an advanced undergraduate course as an introduction to basic C. elegans techniques and neuromuscular function. In a more advanced discovery-based course, students can use this assay to determine if the loss of C. elegans homologs of genes mutated in individuals with myasthenic syndromes, muscular dystrophies, or myopathies alter levamisole sensitivity. In conclusion, this simple, inexpensive levamisole sensitivity assay provides a hands-on approach to learning about signaling at the NMJ and gaining experience working with the C. elegans model system.

Activation of postsynaptic acetylcholine receptors (AChRs) on skeletal muscle results in an electrical signal that leads to muscle contraction. Disruption of neuromuscular function results in myasthenic syndromes and muscular dystrophies in humans1,2,3,4. The nematode Caenorhabditis elegans has been used extensively to learn about evolutionarily conserved fundamental biological processes and mechanisms of disease. Vertebrate skeletal muscles and C. elegans body wall muscles are functionally equivalent in the control of locomotion5. Here, a simple assay is presented that can be used to compare wild-type C. elegans with mutants that have altered neuromuscular signaling or muscle function.
Excitatory and inhibitory inputs received from cholinergic and GABAergic motor neurons, respectively, cause muscles on one side of the C. elegans body to contract while the muscles on the other side relax, enabling coordinated locomotion6. Levamisole, an anthelmintic agent used to treat parasitic nematode infections, binds to and constitutively activates one class of AChRs on the body wall muscles, resulting in time-dependent paralysis7. Thus, altered sensitivity to levamisole can be used to identify C. elegans with defects in the balance of excitatory and inhibitory signaling7,8,9,10,11,12,13,14,15. For example, mutations in subunits of the levamisole-sensitive AChR (L-AChR), such as UNC-63, as well as the C. elegans homolog of Cubilin, LEV-10, which is required for L-AChR clustering at the synapse, impact excitatory signaling and result in levamisole resistance7,8. Mutations in the GABAA receptor UNC-49 reduce inhibitory signaling and cause hypersensitivity to levamisole12.
Hypersensitivity or resistance to levamisole has been traditionally assessed by transferring animals to agar plates containing levamisole and then regularly prodding the worms to determine the time point at which paralysis occurs13,14,15. We have developed a liquid levamisole swim assay that eliminates the need for physical manipulation of the animals and allows for the screening of hundreds of animals in just 1 h. Here, the use of this assay with wild-type, unc-63(x26), lev-10(x17), and unc-49(e407) animals is described. However, this protocol can also be performed on C. elegans exposed to RNAi, as was done to validate knockdowns&#160;identified in a genome-wide RNAi screen for altered levamisole sensitivity11.
For this pharmacological assay, worms were grown to adulthood in 24-well plates, 0.4 mM levamisole in M9 buffer was added to each well, and the number of moving animals was recorded every 5 min for 1 h. Levamisole-induced paralysis was visually observed over time, and after the completion of the assay, data were quantified. The evaluation of mutants along with wild-type C. elegans allows students to first make predictions about potential phenotypic effects and then perform experiments to test their hypotheses. In conclusion, this simple, inexpensive, liquid levamisole assay is an ideal way to demonstrate the impact of the loss of specific genes on NMJ function.

<table><tbody><tr><td>60 mm non-vented, sharp edge Petri Dishes</td><td>TriTech Research</td><td>Cat #: T3308</td><td></td></tr><tr><td>BD Difco Bacto Agar</td><td>Fisher Scientific</td><td>Cat#: DF0140-01-0</td><td></td></tr><tr><td>BioLite 24 Well Multidish</td><td>Fisher Scientific</td><td>Cat#:12-556-006</td><td></td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Fisher Scientific</td><td>Cat#: BP510-500</td><td></td></tr><tr><td>Cholesterol</td><td>MP Biomedicals</td><td>Cat#: 02101382-CF</td><td></td></tr><tr><td>&lt;em&gt;eri-1(mg366)&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td>GR1373</td><td></td></tr><tr><td>Gibco Bacto Peptone</td><td>Fisher Scientific</td><td>Cat#: DF0118-17-0</td><td></td></tr><tr><td>Gibco Bacto Tryptone</td><td>Fisher Scientific</td><td>Cat#: DF0123-17-3</td><td></td></tr><tr><td>GraphPad Prism (Statistical software)</td><td>GraphPad Software, Inc.</td><td></td><td></td></tr><tr><td>&lt;em&gt;lev-10(x17)&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td>ZZ17</td><td></td></tr><tr><td>Levamisole hydrochloride</td><td>Fisher Scientific</td><td>Cat#: AC187870100</td><td></td></tr><tr><td>Low Splash Regular Bleach - 121oz - up &amp;amp; up</td><td>Target</td><td>Search target.com</td><td></td></tr><tr><td>Magnesium Sulfate Heptahydrate</td><td>Fisher Scientific</td><td>Cat#: BP213-1</td><td></td></tr><tr><td>Microsoft Excel</td><td>Microsoft, Inc</td><td></td><td></td></tr><tr><td>N2 wild type</td><td>Caenorhabditis Genetics Center (CGC)</td><td>Bristol N2</td><td></td></tr><tr><td>OP50 &lt;em&gt;E. coli&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td>OP50</td><td></td></tr><tr><td>Potassium Phosphate Dibasic</td><td>Fisher Scientific</td><td>Cat#: BP363-1</td><td></td></tr><tr><td>Potassium Phosphate Monobasic</td><td>Fisher Scientific</td><td>Cat#: BP362-500</td><td></td></tr><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>Cat#: BP358-1</td><td></td></tr><tr><td>Sodium Hydroxide 10N</td><td>Fisher Scientific</td><td>Cat#: SS255-1</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Fisher Scientific</td><td>Cat#: BP332-1</td><td></td></tr><tr><td>&lt;em&gt;unc-49(e407)&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td>CB407</td><td></td></tr><tr><td>&lt;em&gt;unc-63(x26)&lt;/em&gt;</td><td>Caenorhabditis Genetics Center (CGC)</td><td>ZZ26 </td><td></td></tr></tbody></table>

measuring caenorhabditis elegans sensitivity to the acetylcholine receptor agonist levamisole

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle contraction. As in vertebrate skeletal muscle, cholinergic signaling in the body wall muscles of the model organism Caenorhabditis elegans is required for locomotion. Exposure to levamisole, a pharmacological agonist of one class of ACh receptors on the body wall muscles, causes time-dependent paralysis of wild-type animals. Altered sensitivity to levamisole suggests defects in signaling at the NMJ or muscle function. Here, a protocol for a liquid levamisole assay performed on C. elegans grown in 24-well plates is presented. Vigorous swimming of the animals in liquid allows for the assessment and quantitation of levamisole-induced paralysis in hundreds of worms over a one-hour time period without requiring physical manipulation. This procedure can be used with both wild-type and mutants that have altered sensitivity to levamisole to demonstrate the functional consequences of altered signaling at the NMJ.

Signaling through AChRs and GABA receptors allows muscles on one side of the C. elegans body to contract while the muscles on the other side relax, enabling coordinated locomotion6,16. Levamisole binding to ionotropic L-AChRs causes contraction, leading to time-dependent paralysis of wild-type animals.&#160;Here, we assessed the sensitivity of wild type, unc-63 L-AChR mutant, lev-10 L-AChR clustering mutant, and unc-49 GABAA receptor mutant C. elegans to levamisole. Mutations in subunits of the L-AChR, as well as in genes required for trafficking L-AChRs to the muscle plasma membrane, clustering of postsynaptic L-AChRs, and downstream Ca2+ signaling, cause resistance to levamisole-induced paralysis7,8,9,10,11,16,17, as observed in the unc-63 and lev-10 mutants (Figure 2B,C). Loss of the GABA-gated ion channel UNC-49 caused levamisole hypersensitivity (Figure 2B,C) due to disruption of the proper balance of cholinergic and GABAergic signaling12. When this assay was recently performed by undergraduates in an advanced genetics laboratory, 100% of students observed levamisole resistance with the unc-63 and lev-10 mutants, while 88% observed levamisole hypersensitivity with the unc-49 mutant. These data collected with the liquid levamisole swim assay are consistent with phenotypes observed in traditional plate-based levamisole assays7,8,11,12,13.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64056/64056fig01.jpg" /> 
Figure 1: Schematic of levamisole assay preparation and implementation. 24-well NGM plates are poured; 2 days later, OP50 Escherichia coli is seeded into the wells. C. elegans are synchronized through bleach prep, and first larval stage (L1) animals for each strain to be assayed (wild type, black; unc-63(x26), magenta; lev-10(x17), green; unc-49(e407), blue) are pipetted into the wells according to a predetermined plate map the following day. After 3 days of growth at 20 &#186;C, or when the animals reach adulthood, 0.4 mM levamisole in M9 buffer is added to each well, and the number of moving animals is counted every 5 min for 1 h. <a href="https://www.jove.com/files/ftp_upload/64056/64056fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64056/64056fig02.jpg" /> 
Figure 2: Levamisole-induced paralysis in wild type and representative mutant C. elegans. (A) Exposure to 0.4 mM levamisole causes time-dependent paralysis; this is not observed for animals swimming in 1x M9 buffer for 1 h. (B) Time-dependent paralysis of wild-type (black), unc-63(x26) (magenta), lev-10(x17) (green), and unc-49(e407) (blue) animals in the levamisole swim assay. Loss of the L-AChR subunit UNC-63 or L-AChR clustering protein LEV-10&#160;resulted in levamisole resistance, and loss of the GABAA receptor UNC-49 caused levamisole hypersensitivity. n &#8805; 50 per genotype, p &#60; 0.0001 for all mutants compared to the wild type in this representative experiment. (C) Using the same data as in panel (B), the percentage of moving animals at each time point (average of the percentages for each well &#177; SE) was determined for each strain. <a href="https://www.jove.com/files/ftp_upload/64056/64056fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole at JoVE.com

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

The present protocol describes an assay to determine response to levamisole, a pharmacological agonist of one class of Caenorhabditis elegans acetylcholine receptors. In this liquid levamisole swim assay, researchers visually observe and quantitate the time-dependent paralysis of animals cultivated in 24-well plates.

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

At the neuromuscular junction (NMJ), the binding of the excitatory neurotransmitter acetylcholine (ACh) to postsynaptic receptors leads to muscle ...

measuring-caenorhabditis-elegans-sensitivity-to-acetylcholine

Research

JoVE Journal

Genetics

3.7K Views.  University of Delaware. The present protocol describes an assay to determine response to levamisole, a pharmacological agonist of one class of Caenorhabditis elegans acetylcholine receptors. In this liquid levamisole swim assay, researchers visually observe and quantitate the time-dependent paralysis of animals cultivated in 24-well plates. 

Video: Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

Assaying &beta;-amyloid Toxicity using a Transgenic C. elegans Model

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant mechanism(s) of toxicity are still unclear. A&beta; is also deposited intramuscularly in Inclusion Body Myositis, a severe human myopathy. The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express human A&beta;. Depending on the tissue or timing of A&beta; expression, transgenic worms can have readily measurable phenotypes that serve as a read-out of A&beta; toxicity. For example, transgenic worms with pan-neuronal A&beta; expression have defects is associative learning (Dosanjh et al. 2009), while transgenic worms with constitutive muscle-specific expression show a progressive, age-dependent paralysis phenotype (Link, 1995; Cohen et al. 2006). One particularly useful C. elegans model employs a temperature-sensitive mutation in the mRNA surveillance system to engineer temperature-inducible muscle expression of an A&beta; transgene, resulting in a reproducible paralysis phenotype upon temperature upshift (Link et al. 2003). Treatments that counter A&beta; toxicity in this model [e.g., expression of a protective transgene (Hassan et al. 2009) or exposure to Ginkgo biloba extracts (Wu et al. 2006)] reproducibly alter the rate of paralysis induced by temperature upshift of these transgenic worms. Here we describe our protocol for measuring the rate of paralysis in this transgenic C. elegans model, with particular attention to experimental variables that can influence this measurement.

Assaying &#946;-amyloid Toxicity using a Transgenic C. elegans Model

The intensely studied nematode worm Caenorhabditis elegans can be transgenically engineered to express the human &beta;-amyloid peptide (A&beta;). Induced expression of A&beta; in C. elegans muscle leads to a rapid, reproducible paralysis phenotype that can be used to monitor treatments that modulate A&beta; toxicity.

Accumulation of the &beta;-amyloid peptide (A&beta;) is generally believed to be central to the induction of Alzheimer's disease, but the relevant ...

Neuroscience

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 ...

Biology

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

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Biochemistry

Measuring Caenorhabditis elegans Life Span on Solid Media

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode Caenorhabditis elegans has emerged as a principal model used to study the biology of aging. Because virtually every biological subsystem undergoes functional decline with increasing age, life span is the primary endpoint of interest when considering total rate of aging. In nematodes, life span is typically defined as the number of days an animal remains responsive to external stimuli. Nematodes can be propagated either in liquid media or on solid media in plates, and techniques have been developed for measuring life span under both conditions. Here we present a generalized protocol for measuring life span of nematodes maintained on solid nematode growth media and fed a diet of UV-killed bacteria. These procedures can easily be adapted to assay life span under various common conditions, including a diet consisting of live bacteria, dietary restriction, and RNA interference.

Measuring Caenorhabditis elegans Life Span on Solid Media

In this article we present a general protocol for measuring life span of nematodes maintained on solid media with UV-killed bacterial food.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The nematode ...

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.

Alcohol use disorders are a significant public health concern, for which there are few effective treatment strategies. One difficulty that has delayed the ...

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.

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their ...

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.

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.

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

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Measuring RAN Peptide Toxicity in C. elegans

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis (ALS) and Huntington&#8217;s disease. Recently, repeat expansion-containing RNA was shown to be the substrate for a novel type of protein translation called repeat-associated non-AUG-dependent (RAN) translation. Unlike canonical translation, RAN translation does not require a start codon and only occurs when repeats exceed a threshold length. Because there is no start codon to determine the reading frame, RAN translation occurs in all reading frames from both sense and antisense RNA templates that contain a repeat expansion sequence. Therefore, RAN translation expands the number of possible disease-associated toxic peptides from one to six. Thus far, RAN translation has been documented in eight different repeat expansion-based neurodegenerative and neuromuscular diseases. In each case, deciphering which RAN products are toxic, as well as their mechanisms of toxicity, is a critical step towards understanding how these peptides contribute to disease pathophysiology. In this paper, we present strategies to measure the toxicity of RAN peptides in the model system C. elegans. First, we describe procedures for measuring RAN peptide toxicity on the growth and motility of developing C. elegans. Second, we detail an assay for measuring postdevelopmental, age-dependent effects of RAN peptides on motility. Finally, we describe a neurotoxicity assay for evaluating the effects of RAN peptides on neuron morphology. These assays provide a broad assessment of RAN peptide toxicity and may be useful for performing large-scale genetic or small molecule screens to identify disease mechanisms or therapies.

Measuring RAN Peptide Toxicity in C. elegans

Repeat-associated non-ATG-dependent translational products are emerging pathogenic features of several repeat expansion-based diseases. The goal of the protocol described is to evaluate toxicity caused by these peptides using behavioral and cellular assays in the model system C. elegans.

C. elegans is commonly used to model age-related neurodegenerative diseases caused by repeat expansion mutations, such as Amyotrophic Lateral Sclerosis ...

Measuring Caenorhabditis elegans Sensitivity to the Acetylcholine Receptor Agonist Levamisole

Summary

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Chapters in this video

Assaying β-amyloid Toxicity using a Transgenic C. elegans Model

Paradigms for Pharmacological Characterization of C. elegans Synaptic Transmission Mutants

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

Measuring Caenorhabditis elegans Life Span on Solid Media

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

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

C. elegans Chemotaxis Assay

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

Measuring RAN Peptide Toxicity in C. elegans