Name: examining the effect of pesticides on caenorhabditis elegans neurons
Uploaded: 2022-05-27T15:00:00
Description: Watch this Scientific Journal Video about Examining the Effect of Pesticides on Caenorhabditis elegans Neurons at JoVE.com

The work described in this manuscript was done for an intermediate-level class in neuroscience. Funds for the reagents and supplies were provided by the Biology Department at Vassar College. The microscopes and digital imaging system were also provided by the Biology Department at Vassar College. The author thanks all the many students who took this course.

All use of invertebrate animals was in compliance with the animal care and use guidelines of the institution.
1. Preparation of pesticide-coated Petri plates
<ol>
	<li>Prepare agar Petri dishes (6 cm diameter work best) using standard procedures6. 
	NOTE: These can be made months in advance and stored in the refrigerator until use. Bring plates to room temperature (RT) on the benchtop. In most cases, the student groups should be provided with a p...

<ol>
	<li>Duzguner, V., Erdogan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+exposure+to+imidacloprid+induces+inflammation+and+oxidative+stress+in+the+liver+&amp;+central+nervous+system+of+rats.">Chronic exposure to imidacloprid induces inflammation and oxidative stress in the liver &amp; central nervous system of rats.</a> Pesticide Biochemistry and Physiology. 104 (1), 58-64 (2012).</li><li>Catae, A. 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=Exposure+to+a+sublethal+concentration+of+imidacloprid+and+the+side+effects+on+target+and+nontarget+organs+of+Apis+mellifera+(Hymenoptera,+Apidae).">Exposure to a sublethal concentration of imidacloprid and the side effects on target and nontarget organs of Apis mellifera (Hymenoptera, Apidae).</a> Ecotoxicology. 27 (2), 109-121 (2018).</li><li>Bradford, B. R., Whidden, E., Gervasio, E. D., Checchi, P. M., Raley-Susman, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonicotinoid-containing+insecticide+disruption+of+growth,+locomotion,+and+fertility+in+Caenorhabditis+elegans.">Neonicotinoid-containing insecticide disruption of growth, locomotion, and fertility in Caenorhabditis elegans.</a> PLOS One. 15 (9), 0238637 (2020).</li><li>Jospin, 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=A+neuronal+acetylcholine+receptor+regulates+the+balance+of+muscle+excitation+and+inhibition+in+Caenorhabditis+elegans.">A neuronal acetylcholine receptor regulates the balance of muscle excitation and inhibition in Caenorhabditis elegans.</a> PLoS Biology. 7 (12), 1000265 (2009).</li><li>Susman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Discovery-Based Learning in the Life Sciences. , (2015).</li><li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+C.+elegans.">Maintenance of C. elegans.</a> WormBook. , (2006).</li><li>Brody, H. A., Chou, E., Gray, J. M., Pokyrwka, N. J., Raley-Susman, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mancozeb-induced+behavioral+deficits+precede+structural+neural+degeneration.">Mancozeb-induced behavioral deficits precede structural neural degeneration.</a> NeuroToxicology. 34, 74-81 (2013).</li><li>Chen, P., Martinez-Finley, E. J., Bornhorst, J., Chakraborty, S., Aschner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metal-induced+neurodegeneration+in+C.+elegans.">Metal-induced neurodegeneration in C. elegans.</a> Frontiers in Aging Neuroscience. 5, (2013).</li><li>Hart, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavior.">Behavior.</a> WormBook. , (2006).</li><li>Harlow, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nematode+Caenorhabditis+elegans+as+a+tool+to+predict+chemical+activity+on+mammalian+development+and+identify+mechanisms+influencing+toxicological+outcome.">The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome.</a> Scientific Reports. 6 (1), 22965 (2016).</li></ol>

The protocols described in this manuscript work successfully alone or as part of a multi-week independent student group project. The protocols are also amenable to stand-alone, one-week exploratory experiences. The nematode strains are cheap and easy to maintain in the research laboratory. Students can readily learn how to pick worms with a worm pick or how to move them by flushing plates with water and allowing them to settle by gravity. The experiments can be performed over the course of a single lab period or over mul...

Caenorhabditis elegans is an excellent model organism for laboratory course training in biological science courses for introductory- and intermediate-level students. This laboratory procedure can be used as part of a multi-week module that explores various effects of commonly used pesticides on C. elegans behavior and cell biology. Students can learn how to design and carry out independent projects that teach them data analysis and presentation skills. This paper focuses on the protocols for exposing C. elegans to pesticide mixtures and then observing and analyzing the effects on neuron morphology.
Lawn chemical pesticide mixtures are widely used for residential and agricultural use and can be purchased at any local garden store. There is increasing concern about the safety of these chemicals for humans and wildlife1,2,3. Students can read the scientific literature and select a pesticide for experimental evaluation and, in so doing, can learn about basic biology and neurobiology, as well as important laboratory skills like experimental design and analysis, and general lab skills like pipetting and serial dilutions, dissecting microscopy, fluorescent microscopy, digital photography, and figure production.
The protocols described in this paper can stand alone in an intermediate-level course in biology or neuroscience or be part of a multi-week module that could also include measurements of behaviors governed by particular groups of neurons. For example, described in this protocol is an assessment of the morphology of cholinergic neurons that govern locomotion using a strain of nematode that expresses GFP (LX 929) in cholinergic neurons4. These strains can be obtained for very low prices from the Caenorhabditis elegans Genetics Center (https://cgc.umn.edu/). Strains expressing GFP in dopaminergic neurons (OH 7457), cholinergic neurons (LX 929), or mCherry expressed in all neurons (PVX4) are all good choices. Students could also measure locomotion and obtain data to accompany the assessment of morphology. A full description of a multi-week student group project can be found in Susman5.
This student group project is quite inexpensive and easy to set up for groups of four students. The materials needed include a dissecting microscope, access to a fluorescence compound microscope that can have an attached digital camera, Petri plates and access to nematode growth agar, growth-limited bacteria (strain OP50, from the CGC), a gas flame Bunsen burner or an alcohol lamp, an autoclave, platinum wire, and general laboratory supplies like micropipetters, microscope slides, coverslips, and glass Pasteur pipettes. Depending on the chemical toxicant being examined by the student groups, the steps in the protocol might need to occur under a fume hood or with gloves. This protocol uses chemical mixtures that are water-soluble (not volatile), and all safe-handling procedures recommended by the manufacturer are followed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Fisher Scientific</td>
			<td>BP97445</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Fisher Scientific</td>
			<td>MP1AGAH0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol lamp</td>
			<td>Fisher Scientific</td>
			<td>&#160;17012826</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>Fisher Scientific</td>
			<td>17-012-820</td>
			<td></td>
		</tr>
		<tr>
			<td>C. elegans strains</td>
			<td>C. elegans Genetics Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl</td>
			<td>Fisher Scientific</td>
			<td>10035-04-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Fisher Scientific</td>
			<td>AAA1147030</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Nikon</td>
			<td></td>
			<td>These can vary depending on the requirement</td>
		</tr>
		<tr>
			<td>Dissecting scope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli strain (OP50)</td>
			<td>C. elegans Genetics Center</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP2818100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent scope</td>
			<td>Nikon</td>
			<td></td>
			<td>These can vary depending on the requirement</td>
		</tr>
		<tr>
			<td>Imaging software</td>
			<td>Nikon</td>
			<td></td>
			<td>These can vary depending on the requirement</td>
		</tr>
		<tr>
			<td>Inoculation loop</td>
			<td>Fisher Scientific</td>
			<td>&#160;131045</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Broth Base</td>
			<td>Fisher Scientific</td>
			<td>BP9723-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>10034-99-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfuge tubes</td>
			<td>Fisher Scientific</td>
			<td>&#160;05408129</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>Fisher Scientific</td>
			<td>22-265446</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>AS4050</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips</td>
			<td>Fisher Scientific</td>
			<td>94060316</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetters</td>
			<td>Fisher Scientific</td>
			<td>14-386-319</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum wire</td>
			<td>Genesee Scientific</td>
			<td>59-1M30P</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate buffer</td>
			<td>Fisher Scientific</td>
			<td>AAJ61413AP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Fisher Scientific</td>
			<td>AC447810250</td>
			<td></td>
		</tr>
	</tbody>
</table>

examining the effect of pesticides on caenorhabditis elegans neurons

Caenorhabditis elegans is a powerful model organism used in many research laboratories to understand the consequences of exposure to chemical pollutants, pesticides, and a wide variety of toxic substances. These nematodes are easy to work with and can be used to generate novel research findings, even in the undergraduate biology laboratory. A multi-week laboratory series of authentic, student-driven research projects trains students in a toolkit of techniques and approaches in behavioral measurements, cell biology, and microscopy that they then apply to their projects. One technique in that toolkit is quantifying the percentage of neurons exhibiting neurodegenerative damage following exposure to a chemical toxicant like a pesticide. Young adult C. elegans nematodes can be exposed to different concentrations of commercially available pesticides or other types of toxicants for 2-24 h. Then, undergraduate students can visualize different neuron subtypes using fluorescent-expressing strains of C. elegans. These techniques do not require sophisticated image processing software and are effective at even low magnifications, making the need for expensive confocal microscopy unnecessary. This paper demonstrates how to treat the nematodes with pesticides and how to image and score the neurons. It also provides a straightforward protocol for the microscopy and analysis of neuron morphology. The materials used for this technique are inexpensive and readily available in most undergraduate biology departments. This technique can be combined with behavioral measures like locomotion, basal slowing, or egg-laying to conduct a potentially publishable series of experiments and give undergraduate students an authentic research experience at a very low cost.

The methods and protocols described in this paper provide important laboratory skills for intermediate-level undergraduate students in biology or neuroscience. Students can gain important experience in developing an independent project and conducting an experiment of their own design that could provide novel results. Figure 3 shows an optimal result of a student project that eventually became part of a published paper3. While the majority of student projects do not re...

Watch this Scientific Journal Video about Examining the Effect of Pesticides on Caenorhabditis elegans Neurons at JoVE.com

Examining the Effect of Pesticides on Caenorhabditis elegans Neurons

Young adult Caenorhabditis elegans nematodes are exposed to different concentrations of commercial pesticides or other toxicants for 2-24 h. Then, different neurons can be visualized using fluorescent-expressing strains. This paper demonstrates how to expose nematodes to pesticides and assess neuron damage.

Examining the Effect of Pesticides on Caenorhabditis elegans Neurons

Caenorhabditis elegans is a powerful model organism used in many research laboratories to understand the consequences of exposure to chemical pollutants, ...

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