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 plate for each dilution of the pesticide mixture, including a water control.</li>
	<li>Prepare 1 mL of each dilution of the chosen pesticide in labeled 1.5 mL microfuge tubes. For safe handling, be sure to read the instructions and provide gloves, or work in a fume hood, if suggested. 
	NOTE: Suggested dilutions to test: 1 mL of full-strength pesticide, 1:10 dilution in water (100 &#181;L of pesticide, 900 &#181;L of distilled water), 1:100 dilution (10 &#181;L of pesticide, 990 &#181;L of water), 1:1000 dilution (10 &#181;L of the 1:10 dilution, 990 &#181;L of water), etc.</li>
	<li>Make a solution spreader by carefully bending a glass Pasteur pipette (9 ml size) in a Bunsen burner flame. Use the flame to close the opening of the pipette. Then ethanol-sterilize the spreader before each spreading on the Petri plate.</li>
	<li>Using a micropipetter, place 100 &#181;L of the dilution (or water control) on the center of the agar and spread gently across the surface with the sterilized spreader. Re-sterilize the spreader prior to each use.</li>
	<li>Set aside the Petri dishes covered, and let the solution soak into the surface until &#34;dry.&#34; 
	&#8203;NOTE: Depending on the pesticide and humidity levels in the laboratory, this may take some time. Students might need to prepare their plates the day before the exposure period.</li>
	<li>For exposure periods longer than 12 h, add 50 &#181;L of an overnight culture of Escherichia coli to the plates prior to adding the nematodes. For acute experiments (12 h or less), there is no need to have E. coli on the plates.</li>
</ol>
2. Adding nematodes to pesticide-coated plates
NOTE: Students will need to have a plate of adult nematodes (5 day cultures are best) of the fluorescent strain LX929, which is available from the Caenorhabditis elegans Genetics Center. There are two possible procedures for this. If students have had prior experience working with nematodes (for example, if this procedure is part of a multi-week exercise and they have already learned how to pick nematodes with a worm pick), use Step 2.1. If they have not, use Step 2.2.
<ol>
	<li>Pick nematodes with a worm pick. Fashion a worm pick from a 1 in piece of platinum wire that is melted to a Pasteur pipette using a Bunsen burner flame. Pick up a small blob of sticky E. coli onto the pick and then pick up adult nematodes using the sticky blob. For a decent sample size, pick 10 nematodes for each treatment plate. 
	NOTE: Students without much experience can follow Steps 2.2-2.3.</li>
	<li>Use a micropipette to place 1 mL of sterile water onto the plate of nematodes. Swish the water around to lift up the nematodes, then remove the liquid with the nematodes to a 1.5 mL microfuge tube.</li>
	<li>Let the nematodes settle by gravity for about 10 min, then carefully remove at least 500 &#181;L of the water and discard. Resuspend the nematodes and, using a cut off yellow pipette tip, remove 50-100 &#181;L of suspended worms to each treatment plate. Be sure that each plate has at least 10 adult worms.</li>
	<li>Set a timer, or note the time, and expose the nematodes at RT for the chosen time period. During the chosen exposure time, prepare microscope slides for wet mounts. The slides should be made the same day as the microscopy. 
	NOTE: Student groups will have chosen their time of exposure during the planning for their independent study.</li>
</ol>
3. Preparing microscope slides for wet mounts
NOTE: Steps 3.1-3.3 can be performed by each student group. Prepare three slides each.
<ol>
	<li>Prepare two slides by placing a piece of label tape on only one side of the slide (Figure 1, left panel). The tape is used to create the correct thickness for the agarose to form a pad of uniform thickness. Then position a clean, unused slide in between them on the benchtop, as illustrated in the figure.</li>
	<li>Use a micropipetter to place a 10 &#181;L drop of melted agarose (3% in ddH2O, heated using a 65 &#176;C dry block heater) onto the center of the slide that is in the middle.</li>
	<li>Flatten the drop by placing another clean slide on top, perpendicular to the slide with the drop, as shown in the figure (Figure 1, right panel). Flatten by applying pressure, so the agarose drop forms the thickness of the labeling tape.</li>
	<li>The agarose will solidify within about 1 min. Then, apply steady pressure to separate the two slides. The agar circle will adhere to one of the slides. Rest this slide, agar side up, on the benchtop, and allow it to dry for 1-2 min before using.</li>
</ol>
4. Mounting live nematodes to microscope slides
<ol>
	<li>To add nematodes to the prepared slide with the agarose pad, add a 5 &#181;L drop of water containing 1 M sodium azide (NaN3) to the agar pad. This solution anesthetizes the worms and immobilizes them. 
	CAUTION: Sodium azide stock solution can cause illness if ingested.</li>
	<li>Then transfer nematodes (at least 10 per treatment slide) to the droplet using a worm pick. Sterilize the pick before and after using it to transfer nematodes.</li>
	<li>Use forceps to add a coverslip by placing it at an angle and slowly lowering it. Prevent the coverslip from slipping or lowering too quickly by using a pencil or metal probe.</li>
	<li>Place the prepared wet mount on a fluorescent microscope to observe the worms, first under 10x magnification and phase contrast, then under 40x magnification with phase contrast and fluorescent illumination.</li>
</ol>
5. Fluorescent microscopy
NOTE: Student groups should make a wet mount of several worms of each type on separate microscope slides, which they should label appropriately. The instructions that follow are general tips about the use of a standard fluorescence compound microscope that can have an attached trinocular setup, digital camera, and computer system. The setup used for this manuscript is seen in Figure 2. Students should be familiar with the settings of the microscope, including the objectives, types of light filters, ability to switch between bright field and fluorescence light, how to send the light signal to the digital camera, etc.
<ol>
	<li>Place one prepared wet mount at a time on the microscope stage and secure it in place using the microscope stage clips.</li>
	<li>Begin viewing the wet mounts by first viewing with the lowest magnification objective, which is typically 10x, and use bright field or phase contrast to visualize and focus on the nematodes.</li>
	<li>Once a nematode is located in the field of view, focus on it using the fine focus knob on the microscope. Switch the illumination to the fluorescence by turning the dial. Then direct the light to the camera to capture the digital images. Adjust the illumination using the imaging software so that the neurons are brightly illuminated but not oversaturated.</li>
	<li>At some point, obtain a separate image of a ruler at the same magnification as the cell images to provide a magnification scale for their figure. Place a short section of a transparent ruler, or a micrometer slide, onto the microscope stage, focus on it using the fine focus knob, and obtain an image using the imaging software.</li>
	<li>Obtain additional images (from at least 10 separate nematodes) at higher magnifications, if possible. The intense light will likely bleach the area from which it is being imaged, so carefully monitor the imaging and do not allow the light to remain on the same area of the slide for long periods of time. Be sure to name the acquired images to keep track of the nematode and region, as well as the treatment and magnification.</li>
	<li>Make a folder on the computer and move the images into the folder for use in analysis and figure preparation.</li>
</ol>
6. Morphological integrity assessment scale
NOTE: A key cytological characteristic of neurodegenerative damage in neurons is a change to the soma morphology. There are three morphologies that can easily be viewed and counted.
<ol>
	<li>Count the neurons with smooth, uniformly fluorescent processes and non-round cell bodies as normal, as shown in Figure 3.</li>
	<li>Often the soma becomes rounded and swollen-looking compared with healthy, young neurons. Count the neurons that look like this as &#34;rounded.&#34;</li>
	<li>Neurons with long neural processes can exhibit blebbing, where there are rounded puncta or even gaps in the processes. Count the neurons that show these features as &#34;blebbed.&#34;</li>
</ol>
7. Data analysis and figure preparation
<ol>
	<li>If the percentage healthy (no rounding, no blebbing) and the percentage impaired (rounding and/or blebbing) for at least ten nematodes for each treatment were counted (Figure 3), perform statistical analysis using available statistical software.</li>
	<li>Count all of the neurons that are fluorescently tagged, keeping a tally of those exhibiting normal morphology, blebbing morphology, or rounding morphology, and then calculate the percentage of each morphology out of the total count for each nematode analyzed.</li>
	<li>To prepare figures, import the images into an appropriate software like Powerpoint and then add labels and a scale bar based on the ruler or micrometer image acquired.</li>
</ol>

<ol><li>Duzguner, V., Erdogan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+exposure+to+imidacloprid+induces+inflammation+and+oxidative+stress+in+the+liver+%26+central+nervous+system+of+rats%5BTitle%5D)+AND+%22Pesticide+Biochemistry+and+Physiology%22%5BJournal%5D)">Chronic exposure to imidacloprid induces inflammation and oxidative stress in the liver & 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/pubmed?term=((Exposure+to+a+sublethal+concentration+of+imidacloprid+and+the+side+effects+on+target+and+nontarget+organs+of+Apis+mellifera+%28Hymenoptera%2C+Apidae%29%5BTitle%5D)+AND+%22Ecotoxicology%22%5BJournal%5D)">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/pubmed?term=((Neonicotinoid-containing+insecticide+disruption+of+growth%2C+locomotion%2C+and+fertility+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22PLOS+One%22%5BJournal%5D)">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/pubmed?term=((A+neuronal+acetylcholine+receptor+regulates+the+balance+of+muscle+excitation+and+inhibition+in+Caenorhabditis+elegans%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSusman%2C+K+%22Discovery-Based+Learning+in+the+Life+Sciences%22">Discovery-Based Learning in the Life Sciences</a>. , Wiley Blackwell. Hoboken, New Jersey. (2015).</li>
<li>Stiernagle, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Maintenance+of+C.+elegans%5BTitle%5D)+AND+%22WormBook%22%5BJournal%5D)">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/pubmed?term=((Mancozeb-induced+behavioral+deficits+precede+structural+neural+degeneration%5BTitle%5D)+AND+%22NeuroToxicology%22%5BJournal%5D)">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/pubmed?term=((Metal-induced+neurodegeneration+in+C.+elegans%5BTitle%5D)+AND+%22Frontiers+in+Aging+Neuroscience%22%5BJournal%5D)">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/pubmed?term=((Behavior%5BTitle%5D)+AND+%22WormBook%22%5BJournal%5D)">Behavior.</a> WormBook. , (2006).</li>
<li>Harlow, P. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+nematode+Caenorhabditis+elegans+as+a+tool+to+predict+chemical+activity+on+mammalian+development+and+identify+mechanisms+influencing+toxicological+outcome%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">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>

There are no conflicts of interest to disclose.

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 multiple weeks. Students can independently design their experiments, or a procedure can be provided to them. Dissecting microscopes do not have to be expensive. The most expensive item is the fluorescent microscope with a digital camera. This is a limitation for teaching laboratories that do not have access to a fluorescent microscope or digital camera.
If a fluorescent microscope and digital camera and computer imaging system are not available for use in the laboratory, the students could measure specific simple behaviors that are governed by particular neurons. Many behavioral assays are available and can be found in Wormbook9. The behaviors can be assessed using the dissecting microscope.
A critical step in this protocol is preparing the microscope slides for the wet mounts of the nematodes. Another critical step is adding the nematodes to the droplet. A final critical step in the protocol is making sure not to expose the nematodes to the fluorescent illumination for so long that it bleaches the signal. It would be important for the instructor to point out these critical steps so that student groups practice and pay attention closely to the instructions they are being given.
The most exciting aspect of this methodology is that students can design and carry out novel and authentic experiments that could yield publishable results or become the basis for more advanced research, like a senior research project. The procedures can be used to study a variety of pesticides, mixtures of pesticides, and many other different environmental chemicals10, which provides students with a truly relevant research experience and training in experimental design, data analysis, figure preparation, and presentation of results.

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><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>&amp;nbsp;17012826</td><td></td></tr><tr><td>Bunsen burner</td><td>Fisher Scientific</td><td>17-012-820</td><td></td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; 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>&lt;em&gt;E. coli&lt;/em&gt; 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>&amp;nbsp;131045</td><td></td></tr><tr><td>LB Broth Base</td><td>Fisher Scientific</td><td>BP9723-500</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>10034-99-8</td><td></td></tr><tr><td>Microfuge tubes</td><td>Fisher Scientific</td><td>&amp;nbsp;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 result in publishable results, most students are able to create a sophisticated figure and figure legend, and most also are able to obtain cell counts, conduct statistical analysis, and create a figure or data table. Particular neurons govern specific behaviors. Thus, when this procedure is part of a multi-week independent project that includes other measurements like assays of locomotion or chemotaxis, the resulting project yields multiple figures and a class presentation of which students can be proud.
Student groups can choose the type of pesticide they wish to explore. Depending on their literature research on the active ingredient of the pesticide they have selected, the students might evaluate different neurons. As all of the neurons in C. elegans are known and their functions well-described, students can choose specific neuronal types, like dopamine neurons or cholinergic neurons, to study in an independently designed experiment. In the example provided in this manuscript, the students examined a neonicotinoid pesticide and focused on cholinergic neurons using a fluorescent strain of nematode expressing GFP in cholinergic neurons. Another example, shown in Figure 4, shows a more typical result from a student group that examined the effects of a manganese-containing pesticide on dopaminergic neurons using a strain in which dopamine neurons express GFP (OH7457). This particular strain shows a bright signal in dopamine neurons, and the neurons were readily visualized by students. However, over time, the fluorescent signal bleaches if the neurons are exposed to the light for long periods of time, as occurred with this student group.
If the students in the course have had very little experience with fluorescent microscopy, it is a good idea to build in an extra week to give the students a chance to repeat their experiment since their skills will dramatically improve with practice.
A challenging aspect for the laboratory instructor is if there are multiple small groups, each assessing the morphology of different neuron types. The different fluorescent strains are not expensive to obtain or maintain, but there is more effort required to have numerous different fluorescent strains on hand. However, it is possible to limit the types of projects to the available strains and resources. Strains that work very well with undergraduate students include the LX929 and OH7547 strains. PVX4, which is a pan-neuronal expressing mCherry, is also a very easy strain to work with7,8.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63845/63845fig01.jpg" /> 
Figure 1: Diagram of how to prepare microscope slides for wet mounts of C. elegans. The left panel shows the placement of label tape and the droplet of 2%-3% agarose (10 &#181;L). Then, in the right panel, a second slide is placed on top at a 90&#176; angle to flatten the droplet of agarose to the thickness of the tape. Once the agarose has solidified, the slides are carefully separated. The agarose pad will adhere to one of the slides. To prevent drying out, the nematodes are placed into a droplet of buffer containing sodium azide (which paralyzes the nematodes) within an hour of the production of the slide with pad. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63845/63845fig01large.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/63845/63845fig02.jpg" /> 
Figure 2: The fluorescence microscope with a digital camera used in this study. The microscope sits at a table that can accommodate small groups (three to four) of students. The microscope has a digital camera connected by a USB cable to a computer with appropriate digital image capture software. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63845/63845fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63845/63845fig03.jpg" /> 
Figure 3: Evaluation of the effects of a neonicotinoid-containing pesticide on neuron morphology. (A) Untreated cholinergic neurons from the fluorescent strain LX929. (B) Pesticide-exposed (T+S) cholinergic neurons. (C) Percentage of neurons exhibiting blebbing in the two conditions. Error bars are the standard error of the mean. *** indicates p &#60; 0.001 from a one-way ANOVA. A total of 113 neurons from 12 individual nematodes were analyzed. This figure has been modified from Bradford, et al.3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63845/63845fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63845/63845fig04.jpg" /> 
Figure 4: Effects of a manganese-containing pesticide on dopamine neuron morphology. The student group did not provide a scale bar for their figure but took the image at 40x magnification. (A) Putative CEPDL and CEPVL neuronal soma (labeled) in control worms. The intact head neuron somata&#160;and their processes indicate a lack of degeneration. (B) Putative CEPD L&#38;R processes (asterisk) and CEPV L&#38;R neuronal soma (labeled) in acute exposed worms. The soma and processes expressed GFP, but there appears to be a dampening of GFP expression in dorsal processes. (C) Putative CEPDL and CEPVL neuronal soma (labeled) in chronic exposed worms. The soma did not express GFP, and the processes were not visible, indicating significant degeneration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63845/63845fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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, ...

examining-the-effect-of-pesticides-on-caenorhabditis-elegans-neurons

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2.3K Views.  Vassar College. Dit protocol is een eenvoudige en haalbare manier voor niet-gegradueerde studenten om authentiek onderzoek uit te voeren dat kan leiden tot publiceerbare resultaten. Het grote voordeel is dat studenten origineel onderzoek kunnen doen naar vragen die ze zelf beslissen. Maak om te beginnen een oplossingsstrooier door voorzichtig een pasteurpipet van negen milliliter in een Bunsen-brander te buigen en de opening van de pipet te sluiten.Vervolgens steriliseer ethanol de strooier voordat el...

Video: Examining the Effect of Pesticides on Caenorhabditis elegans Neurons

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

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and maintenance issues. On the contrary, the nematode Caenorhabditis elegans (C. elegans) has advantages to make it an ideal choice for toxicity testing: a short lifespan, easy cultivation, and efficient reproduction. Here, we describe a protocol for the automatic phenotypic profiling of C. elegans in a 384-well plate. The nematode worms are cultured in a 384-well plate with liquid medium and chemical treatment, and videos are taken of each well to quantify the chemical influence on 33 worm features. Experimental results demonstrate that the quantified phenotype features can classify and predict the acute toxicity for different chemical compounds and establish a priority list for further traditional chemical toxicity assessment tests in a rodent model.

A High-throughput Assay for the Prediction of Chemical Toxicity by Automated Phenotypic Profiling of Caenorhabditis elegans

A quantitative method has been developed to identify and predict the acute toxicity of chemicals by automatically analyzing the phenotypic profiling of Caenorhabditis elegans. This protocol describes how to treat worms with chemicals in a 384-well plate, capture videos, and quantify toxicological related phenotypes.

Applying toxicity testing of chemicals in higher order organisms, such as mice or rats, is time-consuming and expensive, due to their long lifespan and ...

Environment

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Caenorhabditis elegans is a simple genetic organism amenable to large-scale forward and reverse genetic screens and chemical genetic screens. The C. elegans genome includes potential antipsychotic drug (APD) targets conserved in humans, including genes encoding proteins required for neurotransmitter synthesis and for synaptic structure and function. APD exposure produces developmental delay and/or lethality in nematodes in a concentration-dependent manner. These phenotypes are caused, in part, by APD-induced inhibition of pharyngeal pumping1,2. Thus, the developmental phenotype has a neuromuscular basis, making it useful for pharmacogenetic studies of neuroleptics. Here we demonstrate detailed procedures for testing APD effects on nematode development and pharyngeal pumping. For the developmental assay, synchronized embryos are placed on nematode growth medium (NGM) plates containing APDs, and the stages of developing animals are then scored daily. For the pharyngeal pumping rate assay, staged young adult animals are tested on NGM plates containing APDs. The number of pharyngeal pumps per unit time is recorded, and the pumping rate is calculated. These assays can be used for studying many other types of small molecules or even large molecules.

Methods for Studying the Mechanisms of Action of Antipsychotic Drugs in Caenorhabditis elegans

Approaches for testing the effects of antipsychotic drugs (APDs) in Caenorhabditis elegans are demonstrated. Assays are described for testing drug effects on development and viability and on pharyngeal pumping rate. These methods are also applicable for pharmacogenetic experiments with drug classes other than APDs.

Caenorhabditis elegans is a simple genetic organism amenable to  large-scale forward and reverse genetic screens and chemical genetic screens. The C.  ...

Smartphone-Based Imaging for C. elegans Lawn Avoidance Assay

A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay

When exposed to toxic or pathogenic bacteria, the nematode Caenorhabditis elegans displays a learned lawn avoidance behavior, in which the worms gradually leave their food source and prefer to remain outside the bacterial lawn. The assay is an easy way to test the worms' ability to sense external or internal cues to properly respond to harmful conditions. Though a simple assay, counting is time consuming, particularly with multiple samples, and assay durations that span overnight are inconvenient for researchers. An imaging system that can image many plates over a long period is useful but costly. Here, we describe a smartphone-based imaging method to record lawn avoidance in C. elegans. The method requires only a smartphone and a light emitting diode (LED) light box, to serve as a transmitted light source. Using free time-lapse camera applications, each phone can image up to six plates, with sufficient sharpness and contrast to manually count worms outside the lawn. The resulting movies are processed into 10 s audio video interleave (AVI) files for every hourly time point, then cropped to show each single plate to make them more amenable for counting. This method is a cost-effective way for those looking to examine avoidance defects and can potentially be extended to other C. elegans assays.

Author Spotlight: A Smartphone-Based Imaging Method for C. elegans Lawn Avoidance Assay  

This article describes a simple, low-cost method of recording the lawn avoidance behavior of Caenorhabditis elegans, using readily available items such as a smartphone and a light emitting diode (LED) light box. We also provide a Python script to process the video file into a format more amenable for counting.

When exposed to toxic or pathogenic bacteria, the nematode Caenorhabditis elegans displays a learned lawn avoidance behavior, in which the worms gradually ...

Behavior

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 ...

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its completely mapped nervous system and transparent anatomy, presents an ideal model for understanding real-time neural dynamics using calcium indicators. In combination with microfluidic technologies and experimental designs, calcium-imaging studies using these indicators are performed in both free-moving and trapped animals. However, most previous studies utilizing trapping devices, such as the olfactory chip described in Chronis et al., have devices designed for use in the more common hermaphrodite, as the less common male is both morphologically and structurally dissimilar. An adapted olfactory chip was designed and fabricated for increased efficiency in male neuronal imaging with using young adult animals. A turn was incorporated into the worm loading port to rotate the animals and to allow for the separation of the individual neurons within a bilateral pair in 2D imaging. Worms are exposed to a controlled flow of odorant within the microfluidic device, as described in previous hermaphrodite studies. Calcium transients are then analyzed using the open-source software ImageJ. The procedure described herein should allow for an increased amount of male-based C. elegans calcium imaging studies, deepening our understanding of the mechanisms of sex-specific neuronal signaling.

Using an Adapted Microfluidic Olfactory Chip for the Imaging of Neuronal Activity in Response to Pheromones in Male C. Elegans Head Neurons

The use of an adapted &#34;olfactory chip&#34; for the efficient calcium imaging of C. elegans males is described here. Studies of male exposure to glycerol and a pheromone are also shown.

The use of calcium indicators has greatly enhanced our understanding of neural dynamics and regulation. The nematode Caenorhabditis elegans, with its ...

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.
Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 &#181;m and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

This protocol describes the use of genetically encoded Ca2+ reporters to record changes in neural activity in behaving Caenorhabditis elegans worms. &#8195;

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized ...

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

Medicine

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million people worldwide. Since many details about PD etiology remain unknown, studies investigating genetic and environmental contributors to PD are needed to discover methods of prevention, management, and treatment. Proper characterization of dopaminergic neuronal loss may be relevant not only to PD research, but to other increasingly prevalent neurodegenerative disorders.
There are established genetic and chemical models of dopaminergic neurodegeneration in the Caenorhabditis elegans model system, with easy visualization of neurobiology supported by the nematodes&#39; transparency and invariant neuronal architecture. In particular, hermaphroditic C. elegans&#39; dopaminergic neuron morphological changes can be visualized using strains with fluorescent reporters driven by cell-specific promotors such as the dat-1 dopamine transporter gene, which is expressed exclusively in their eight dopaminergic neurons.
With the capabilities of this model system and the appropriate technology, many laboratories have studied dopaminergic neurodegeneration. However, there is little consistency in the way the data is analyzed and much of the present literature uses binary scoring analyses that capture the presence of degeneration but not the full details of the progression of neuron loss. Here, we introduce a universal scoring system to assess morphological changes and degeneration in C. elegans&#39; cephalic neuron dendrites. This seven-point scale allows for analysis across a full range of dendrite morphology, ranging from healthy neurons to complete dendrite loss, and considering morphological details including kinks, branching, blebs, and breaks. With this scoring system, researchers can quantify subtle age-related changes as well as more dramatic chemical-induced changes. Finally, we provide a practice set of images with commentary that can be used to train, calibrate, and assess the scoring consistency of researchers new to this method. This should improve within- and between- laboratory consistency, increasing rigor and reproducibility.

Quantifying Levels of Dopaminergic Neuron Morphological Alteration and Degeneration in Caenorhabditis elegans

In this article, we showcase how to use a seven-point scoring system to consistently quantify changes to dopaminergic neuron dendrite morphology in C. elegans. This system is intended for analyses of dopaminergic neurodegeneration assays utilizing genetic, chemical, and age-based models of neurodegenerative disorders.

Dopamine neuron loss is involved in the pathology of Parkinson&#39;s Disease (PD), a highly prevalent neurodegenerative disorder affecting over 10 million ...

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

Examining the Effect of Pesticides on Caenorhabditis elegans Neurons

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