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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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Congenital+myopathies+with+secondary+neuromuscular+transmission+defects;+A+case+report+and+review+of+the+literature.">Congenital myopathies with secondary neuromuscular transmission defects; A case report and review of the literature.</a> Neuromuscular Disorders. 24 (12), 1103-1110 (2014).</li><li>Montagnese, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+patients+with+GMPPB+mutation:+The+overlapping+phenotypes+of+limb-girdle+myasthenic+syndrome+and+limb-girdle+muscular+dystrophy+dystroglycanopathy.">Two patients with GMPPB mutation: The overlapping phenotypes of limb-girdle myasthenic syndrome and limb-girdle muscular dystrophy dystroglycanopathy.</a> Muscle and Nerve. 56 (2), 334-340 (2017).</li><li>Gieseler, K., Qadota, H., Benian, G. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>eri-1(mg366)</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>lev-10(x17)</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 &#38; 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 E. coli</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>unc-49(e407)</td>
			<td>Caenorhabditis Genetics Center (CGC)</td>
			<td>CB407</td>
			<td></td>
		</tr>
		<tr>
			<td>unc-63(x26)</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

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged as a powerful tool for gene-expression analysis and transcriptome mapping. However, handling RNA-Seq datasets requires sophisticated computational expertise and poses inherent challenges for biology researchers. This bottleneck has been mitigated by the open access Galaxy project that allows users without bioinformatics skills to analyze RNA-Seq data, and the Database for Annotation, Visualization, and Integrated Discovery (DAVID), a Gene Ontology (GO) term analysis suite that helps derive biological meaning from large data sets. However, for first-time users and bioinformatics' amateurs, self-learning and familiarization with these platforms can be time-consuming and daunting. We describe a straightforward workflow that will help C. elegans researchers to isolate worm RNA, conduct an RNA-Seq experiment and analyze the data using Galaxy and DAVID platforms. This protocol provides stepwise instructions for using the various Galaxy modules for accessing raw NGS data, quality-control checks, alignment, and differential gene expression analysis, guiding the user with parameters at every step to generate a gene list that can be screened for enrichment of gene classes or biological processes using DAVID. Overall, we anticipate that this article will provide information to C. elegans researchers undertaking RNA-Seq experiments for the first time as well as frequent users running a small number of samples.

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Galaxy and DAVID have emerged as popular tools that allow investigators without bioinformatics training to analyze and interpret RNA-Seq data. We describe a protocol for C. elegans researchers to perform RNA-Seq experiments, access and process the dataset using Galaxy and obtain meaningful biological information from the gene lists using DAVID.

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System (REQS)

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel have been described. However, a method to measure the amount of additional animal activity induced through the exercise treatment has been lacking. The Rotating Exercise Quantification System (REQS) fills this need, providing a measure of animal activity for animals experiencing rotational exercise. This protocol details how to use the REQS to assess animal activity during rotational exercise and illustrates the type of data that can be generated. Here, we demonstrate how the REQS is used to measure sex- and strain-specific differences in exercise induced activity. The REQS can also be used to evaluate the impact of various other experimental parameters such as age, diet, or population size on exercise induced activity. In addition, it can be used to compare the efficacy of different exercise training protocols. Importantly, it provides an opportunity to standardize exercise treatments between strains, allowing the researcher to achieve equal amounts of activity between groups if needed. Thus, the REQS is a notable new resource for exercise biologists working with the Drosophila model system and complements existing exercise systems.

Measuring Exercise Levels in Drosophila melanogaster Using the Rotating Exercise Quantification System REQS

The Rotating Exercise Quantification System (REQS) can induce exercise in Drosophila melanogaster through rotation while simultaneously measuring the amount of activity performed by the animals. Here, we present a point-by-point protocol detailing how to measure activity levels of animals experiencing rotational exercise treatments using the REQS.

Drosophila melanogaster is a new model organism for studies in exercise biology. To date, two main exercise systems, the Power Tower and the Treadwheel ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

Measuring Microbial Mutation Rates with the Fluctuation Assay

Fluctuation assays are widely used for estimating mutation rates in microbes growing in liquid environments. Many cultures are each inoculated with a few thousand cells, each sensitive to a selective marker that can be assayed phenotypically. These parallel cultures grow for many generations in the absence of the phenotypic marker. A subset of cultures is used to estimate the total number of cells at risk of mutations (i.e., the population size at the end of the growth period, or Nt). The remaining cultures are plated onto the selective agar. The distribution of observed resistant mutants among parallel cultures is then used to estimate the expected number of mutational events, m, using a mathematical model. Dividing m by Nt gives the estimate of the mutation rate per locus per generation. The assay has three critical aspects: the chosen phenotypic marker, the chosen volume of parallel cultures, and ensuring that the surface on the selective agar is completely dry before the incubation. The assay is relatively inexpensive and only needs standard laboratory equipment. It is also less laborious than alternative approaches, such as mutation accumulation and single-cell assays. The assay works on organisms that go through many generations rapidly and it depends on assumptions about the fitness effects of markers and cell death. However, recently developed tools and theoretical studies mean these issues can now be addressed analytically. The assay allows mutation rate estimation of different phenotypic markers in cells with different genotypes growing in isolation or in a community. By conducting multiple assays in parallel, assays can be used to study how an organism&#39;s environmental context affects spontaneous mutation rate, which is crucial for understanding antimicrobial resistance, carcinogenesis, aging, and evolution.

Here, a protocol is presented to perform a fluctuation assay and estimate microbial mutation rate using phenotypic markers. This protocol will enable researchers to assay mutations in diverse microbes and environments, determining how genotype and ecological context affect spontaneous mutation rates.

Fluctuation assays are widely used for estimating mutation rates in microbes growing in liquid environments. Many cultures are each inoculated with a few ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...