Name: aversive associative learning and memory formation by pairing two chemicals in caenorhabditis elegans
Uploaded: 2022-06-23T15:00:00.000+00:00
Duration: 7 min 17 s
Description: Watch this Scientific Journal Video about Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans at JoVE.com

We are grateful to Takashi Murayama, Ei-ichiro Saita, Iou Ven Chang, and Hitomi Ohtaki for their technical assistance and comments on the manuscript. Strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). This work was supported by funding from Okinawa Institute of Science and Technology Graduate University.

1. Recipes
<ol>
	<li>NGM agar plates (step 2.1.)
	<ol>
		<li>To prepare 6 cm NGM plates, dissolve 2.5 g of peptone, 3 g of NaCl, and 17 g of agar in 850 mL of doubly deionized H2O (ddH2O). Bring the total volume to 972 mL with ddH2O.</li>
		<li>After autoclaving, cool down to ~65 &#176;C and add 1 mL of 5 mg/mL cholesterol dissolved in ethanol, 1 mL each of 1 M CaCl2 and 1 M MgSO4, and 25 mL of 1 M potassium phosphate (pH 6.0). After mixing well, dispense 8 mL each to 6 cm (in diameter) Petri dishes.</li>
		<li>Keep the plates with lids on a bench at room temperature (RT) for 1 day, and then keep them in plasticware in a cold room until use.</li>
	</ol>
	</li>
	<li>Prepare Luria-Bertani (LB) medium (step 2.1.) by dissolving 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl in 1 L of ddH2O. Adjust the pH to 7.0 with 5 N NaOH (several drops) and sterilize by autoclaving.
	<ol>
		<li>Prepare LB plates by adding 15 g of agar to LB medium. After autoclaving, cool down to ~60 &#176;C and dispense 12 mL each to 9 cm (in diameter) Petri dishes. Keep plates in plasticware in a cold room until use.</li>
	</ol>
	</li>
	<li>To make an animal collector (step 2.4.), attach nylon mesh (30 &#181;m mesh size) to the bottom of a clear acrylic cylindrical pipe (3.5 cm in length, 3 cm in external diameter, 2 mm in wall thickness) with glue.</li>
	<li>To make 0.25% aqueous gelatin solution (step 2.4.), dissolve 0.25 g of gelatin in 100 mL of ddH2O. Sterilize by autoclaving.</li>
	<li>Chemotaxis assay plates (step 5.1.)
	<ol>
		<li>To make agar plates for chemotaxis assay, dissolve 15 g of agar in 993 mL of ddH2O by autoclaving, and cool down the solution to ~65 &#176;C.</li>
		<li>Then, add 5 mL of autoclaved 1 M potassium phosphate (pH 6.0), 1 mL of 1 M CaCl2, and&#160;1 mL of 1 M MgSO4 to the agar solution. All of these solutions are separately sterilized by autoclaving.</li>
		<li>Dispense 10 mL of the mixed solution to a 6 cm Petri dish. Place these plates with lids on a bench at RT for two days, and then put them on wet paper towels in plasticware at RT until use. These plates can be used for up to 10 days.</li>
	</ol>
	</li>
	<li>To make chemotaxis assay buffer (step 5.4.), mix 5 mL of 1 M potassium phosphate (pH 6.0), 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, and 993 mL of ddH2O. Separately sterilize all these solutions by autoclaving.</li>
	<li>To make a 40 mL CS/US mixture solution (1% aqueous 1-propanol and HCl [pH 4.0]) (step 3.1. and step 4.1.), add 0.4 mL of absolute 1-propanol and 4 &#181;L of 5 M HCl (0.1 mM at final concentration) to 39.6 mL of ddH2O. Keep the solution at RT.</li>
	<li>To make ddH2O, treat tap water 2x with water purification systems (refer to Table of Materials).</li>
	<li>Prepare a liquid culture (OD600 = ~0.7) of Escherichia coli OP50 by inoculating a fresh colony with a toothpick into 10 mL of LB medium and incubating at 37 &#176;C for 7-8 h. Bacteria cultivated for a longer period can affect the outcome of conditioning, perhaps due to secondary metabolites.</li>
</ol>
2. Preparation of synchronized&#160;&#160;C.&#160; elegans
<ol>
	<li>Using standard methods26, cultivate animals on 6 cm NGM plates (step 1.1.). NGM plates are prepared by spreading 0.2 mL of an E. coli OP50 liquid culture in LB medium (see step 1.9.) and incubating at RT for no more than 24 h (old bacteria can affect the outcome of conditioning). 
	NOTE: C. elegans learning and memory are extremely sensitive to mechanical, chemical, and temperature stresses. Therefore, it is highly recommended to cultivate animals, maintain all reagents including water, and perform all assays at RT between 17 &#176;C and 20 &#176;C. Physical and mechanical stimulation such as vortexing, rough pipetting, and centrifugation must be avoided. Fresh 1-propanol should be used every 3 months at the longest for an unknown reason. Importantly, animals must be cultivated with plenty of food as starvation can seriously affect the outcome of conditioning.</li>
	<li>On day 1, pick and place five well-fed gravid animals (put more mutant animals that lay eggs slowly) on each of the four 6 cm NGM plates with a platinum worm picker, and let them lay ~50 eggs for 3 h at RT to obtain a synchronized population of adult animals. Stop egg laying by removing parent animals from the plates with a platinum worm picker. 
	NOTE: Seeded plates should be kept at RT to minimize stress to the animals.</li>
	<li>Cultivate the animals at RT for about 5 days, which is the time it takes for animals to reach their mature adult stage, not the young adult stage. 
	NOTE: The cultivation period between 4.5 days and 5.5 days should be adjusted depending on the conditions since younger adult animals are more sensitive to the chemicals used for conditioning than mature adult animals (Supplemental Figure 1). After conditioning, younger adult animals may show lower chemotaxis index (C.I.) values.</li>
	<li>Collect ~200 adult animals in an animal collector (see step 1.4.) by washing each plate with 1 mL of 0.25% aqueous gelatin (step 1.4.) This aqueous gelatin prevents adhesion of the animals to the surface of plastics such as pipette tips.</li>
	<li>Wash the animals in the collector with ddH2O (step 1.8.) by very gently moving the collector up and down 2x in ~10 mL of ddH2O. Repeat this process 2x more (3x in total) with ~10 mL of ddH2O each to prevent bacterial contamination. 
	&#8203;NOTE: Bacterial contamination seriously affects the chemotaxis of animals.</li>
</ol>
3. Mass training for short-term associative learning and memory
NOTE: See Figure 1 for the massed training workflow.
<ol>
	<li>Gently immerse the animal collector containing ~200 animals in 40 mL of a mixture of 1% 1-propanol and HCl (pH 4.0 after mixing with 1-propanol; see step 1.7.) in a crystallizing dish for ~1 s. 
	NOTE: For the control animals, do the same but dip only in 1% aqueous 1-propanol. It would be better to treat animals with HCl (pH 4.0) only as another control.</li>
	<li>Wash the animals in the collector by very gently immersing the collector 1x in 10 mL of ddH2O in a well of a 6-well tissue culture plate. 
	NOTE: This washing step must be very gentle and only be done 1x as extensive washing may prevent learning.</li>
	<li>Repeat steps 3.1. and 3.2. 10x without interruption (inter-trial interval [ITI], 0 min). 
	&#8203;NOTE: Use fresh ddH2O each time at RT.</li>
	<li>Place the collector on an E. coli OP50 lawn on a 6 cm NGM plate for 10 min at RT in order for the animals to rest.</li>
	<li>Wash the animals in the collector with ddH2O by very gently moving the collector up and down 2x in ~10 mL of ddH2O. Repeat this process 2x more (3x in total) with ~10 mL of ddH2O each to prevent bacterial contamination.</li>
	<li>Proceed to chemotaxis assay as described below (step 5).</li>
</ol>
4. Spaced training for long-term associative learning and memory
NOTE: See Figure 2 for spaced training workflow.
<ol>
	<li>Gently immerse an animal collector containing ~200 animals in 40 mL of a mixture of 1% 1-propanol and HCl (pH 4.0 after mixing with 1-propanol; see step 1.7.) in a crystallizing dish for ~1.0 s. 
	NOTE: Do the same with 1% aqueous 1-propanol only as a control. It would be better to treat animals with HCl (pH 4.0) only as another control.</li>
	<li>Wash the animals in the collector by very briefly immersing the collector 1x in 10 mL of ddH2O in a well of a 6-well tissue culture plate. 
	NOTE: This washing must be very brief as extensive washing may prevent learning.</li>
	<li>Place the collector on an E. coli OP50 lawn on an NGM agar plate in a 6 cm (in diameter) Petri dish for 10 min at RT in order for the animals to rest. 
	NOTE: This resting for 10 min as ITI&#160;is crucial for the animals to consolidate memories for the formation of LTM.</li>
	<li>Repeat steps 4.1.-4.3. 10x.</li>
	<li>Wash the animals in the collector with ddH2O by very gently moving the collector up and down 2x in ~10 mL of ddH2O, which is kept at RT. Repeat this process 2x more (3x in total) with ~10 mL of ddH2O each to prevent bacterial contamination.</li>
	<li>Proceed to chemotaxis assay as described below (step 5.).</li>
</ol>
5. Chemotaxis assay
<ol>
	<li>Prepare agar plates for chemotaxis assay in 6 cm plastic Petri dishes (see step 1.5.).</li>
	<li>Transfer animals in the collector (step 2.5.), which is placed on a flat surface of a plastic Petri dish lid, to a 2 mL microcentrifuge tube with 1 mL of 0.25% aqueous gelatin using a sawed-off pipette tip with a &#62;1 mm (inner diameter) opening. 
	NOTE: It is important to use a sawed-off pipette tip to minimize shear stress on animals.</li>
	<li>Remove the supernatant from the tube after the animals settle at the bottom of the tube by gravity for ~1 min (do not centrifuge).</li>
	<li>Gently resuspend the animals in 1 mL of chemotaxis assay buffer (see step 1.6.) and let them settle down by gravity to the bottom of the tube for ~1 min (do not centrifuge). Remove as much supernatant as possible by pipetting.</li>
	<li>Meantime, diagonally spot 4 &#181;L each of 5% aqueous 1-propanol at two places and spot 4 &#181;L each of ddH2O at two other places in the same way, as shown in Figure 3A. For chemotaxis assay of mutants that have lower sensitivity to 1-propanol, spot higher concentrations of aqueous 1-propanol that result in ~0.6 chemotaxis index (C.I.) values of na&#239;ve mutants, as shown in Supplemental Table 1. 
	NOTE: It is important to complete the spotting procedures as quickly as possible. Spot 5% aqueous 1-propanol since 1% aqueous 1-propanol is too weak to attract animals in the chemotaxis assay. In contrast, use 1% aqueous 1-propanol for conditioning since animals treated with higher concentrations of aqueous 1-propanol than 1% show lower C.I. values.</li>
	<li>Spot 6 &#181;L portions of the animal suspension in chemotaxis assay buffer (step 5.4.) containing ~60 animals at the center of three plates for chemotaxis assay using a sawed-off pipette tip with a ~1.0 mm (inner diameter) opening. Remove liquid as much as possible with a laboratory tissue wick without touching the animals and place a lid on the plate. 
	NOTE: It is important to complete these procedures as quickly as possible.</li>
	<li>Allow the animals to move freely on the plate for 10 min at RT, and then transfer the plate into a glass Petri dish on ice for 3 min to stop chemotaxis. Then, keep the plate in a refrigerator until counting the number of animals on the plate.</li>
	<li>Count the number of animals in four sections, except for those in the center circle, under a stereomicroscope and calculate the chemotaxis index (C.I.) using the equation shown in Figure 3B. From the C.I. values, calculate learning index (L.I.) values as the difference between the C.I. value of the reference animals and the C.I. value of the conditioned animals (L.I. = C.I.reference- C.I.conditioned). 
	NOTE: The C.I. value of the reference animals (C.I.reference) is the mean value of the C.I. values of animals conditioned with 1% aqueous 1-propanol only.</li>
</ol>

<ol>
	<li>White, J. G., Southgate, E., Thomson, J. N., Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+nervous+system+of+the+nematode+Caenorhabditis+elegans.">The structure of the nervous system of the nematode Caenorhabditis elegans.</a> Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 314 (1165), 1 (1986).</li><li>Cook, S. J., 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=Whole-animal+connectomes+of+both+Caenorhabditis+elegans+sexes.">Whole-animal connectomes of both Caenorhabditis elegans sexes.</a> Nature. 571 (7763), 63-71 (2019).</li><li>Witvliet, D., 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=Connectomes+across+development+reveal+principles+of+brain+maturation.">Connectomes across development reveal principles of brain maturation.</a> Nature. 596, 257-261 (2021).</li><li>Hedgecock, E. M., Russell, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+and+mutant+thermotaxis+in+the+nematode+Caenorhabditis+elegans.">Normal and mutant thermotaxis in the nematode Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 72 (10), 4061-4065 (1975).</li><li>Mohri, A., 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=Genetic+control+of+temperature+preference+in+the+nematode+Caenorhabditis+elegans.">Genetic control of temperature preference in the nematode Caenorhabditis elegans.</a> Genetics. 169 (3), 1437-1450 (2005).</li><li>Wen, J. Y. 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=Mutations+that+prevent+associative+learning+in+C.+elegans.">Mutations that prevent associative learning in C. elegans.</a> Behavioral Neuroscience. 111 (2), 354-368 (1997).</li><li>Saeki, S., Yamamoto, M., Iino, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+chemotaxis+revealed+by+paired+presentation+of+a+chemoattractant+and+starvation+in+the+nematode+Caenorhabditis+elegans.">Plasticity of chemotaxis revealed by paired presentation of a chemoattractant and starvation in the nematode Caenorhabditis elegans.</a> Journal of Experimental Biology. 204 (10), 1757-1764 (2001).</li><li>Tomioka, 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=The+insulin/PI3-kinase+pathway+regulates+salt+chemotaxis+learning+in+Caenorhabditis+elegans.">The insulin/PI3-kinase pathway regulates salt chemotaxis learning in Caenorhabditis elegans.</a> Neuron. 51 (5), 613-625 (2006).</li><li>Torayama, I., Ishihara, T., Katsura, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+integrates+the+signals+of+butanone+and+food+to+enhance+chemotaxis+to+butanone.">Caenorhabditis elegans integrates the signals of butanone and food to enhance chemotaxis to butanone.</a> Journal of Neuroscience. 27 (4), 741-750 (2007).</li><li>Kaufman, A. L., Ashraf, J. M., Corces-Zimmerman, M. R., Landis, J. N., Murphy, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+signaling+and+dietary+restriction+differentially+influence+the+decline+of+learning+and+memory+with+age.">Insulin signaling and dietary restriction differentially influence the decline of learning and memory with age.</a> PLoS Biology. 8 (5), 1000372 (2010).</li><li>Stein, G. M., Murphy, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+positive+olfactory+associative+memory+is+a+molecularly+conserved+behavioral+paradigm.">C. elegans positive olfactory associative memory is a molecularly conserved behavioral paradigm.</a> Neurobiology of Learning and Memory. 115, 86-94 (2014).</li><li>Rahmani, A., Chew, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+the+molecular+mechanisms+of+learning+and+memory+using+Caenorhabditis+elegans.">Investigating the molecular mechanisms of learning and memory using Caenorhabditis elegans.</a> Journal of Neurochemistry. 159 (3), 417-451 (2021).</li><li>Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensation+in+C.+elegans.">Chemosensation in C. elegans.</a> WormBook. 25, 1-29 (2006).</li><li>Gray, J. M., Hill, J. J., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+circuit+for+navigation+in+Caenorhabditis+elegans.">A circuit for navigation in Caenorhabditis elegans.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (9), 3184-3191 (2005).</li><li>Cho, C. E., Brueggemann, C., L'Etoile, N. D., Bargmann, C. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+encoding+of+sensory+history+and+behavioral+preference+during+Caenorhabditis+elegans+olfactory+learning.">Parallel encoding of sensory history and behavioral preference during Caenorhabditis elegans olfactory learning.</a> eLife. 5, 14000 (2016).</li><li>Juang, B. T., 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=Endogenous+nuclear+RNAi+mediates+behavioral+adaptation+to+odor.">Endogenous nuclear RNAi mediates behavioral adaptation to odor.</a> Cell. 154 (5), 1010-1022 (2013).</li><li>Neal, S. J., 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=Feeding+state-dependent+regulation+of+developmental+plasticity+via+CaMKI+and+neuroendocrine+signaling.">Feeding state-dependent regulation of developmental plasticity via CaMKI and neuroendocrine signaling.</a> eLife. 4, 10110 (2015).</li><li>Castellucci, V. F., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantal+analysis+of+the+synaptic+depression+underlying+habituation+of+the+gill-withdrawal+reflex+in+Aplysia.">A quantal analysis of the synaptic depression underlying habituation of the gill-withdrawal reflex in Aplysia.</a> Proceedings of the National Academy of Sciences of the United States of America. 71 (12), 5004-5008 (1974).</li><li>Klein, M., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+calcium+current+modulation+underlying+presynaptic+facilitation+and+behavioral+sensitization+in+Aplysia.">Mechanism of calcium current modulation underlying presynaptic facilitation and behavioral sensitization in Aplysia.</a> Proceedings of the National Academy of Sciences of the United States of America. 77 (11), 6912-6916 (1980).</li><li>Amano, H., Maruyama, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aversive+olfactory+learning+and+associative+long-term+memory+in+Caenorhabditis+elegans.">Aversive olfactory learning and associative long-term memory in Caenorhabditis elegans.</a> Learning &amp; Memory. 18 (10), 654-665 (2011).</li><li>Nishijima, S., Maruyama, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appetitive+olfactory+learning+and+long-term+associative+memory+in+Caenorhabditis+elegans.">Appetitive olfactory learning and long-term associative memory in Caenorhabditis elegans.</a> Frontiers in Behavioral Neuroscience. 11, 80 (2017).</li><li>Morrison, G. E., Wen, J. Y. M., Runciman, S., vander Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+associative+learning+in+Caenorhabditis+elegans+is+impaired+in+lrn-1+and+lrn-2+mutants.">Olfactory associative learning in Caenorhabditis elegans is impaired in lrn-1 and lrn-2 mutants.</a> Behavioral Neuroscience. 113 (2), 358-367 (1999).</li><li>Morrison, G. E., vander Kooy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+in+the+AMPA-type+glutamate+receptor,+glr-1,+blocks+olfactory+associative+and+nonassociative+learning+in+Caenorhabditis+elegans.">A mutation in the AMPA-type glutamate receptor, glr-1, blocks olfactory associative and nonassociative learning in Caenorhabditis elegans.</a> Behavioral Neuroscience. 115 (3), 640-649 (2001).</li><li>Bargmann, C. I., Hartwieg, E., Horvitz, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Odorant-selective+genes+and+neurons+mediate+olfaction+in+C.+elegans.">Odorant-selective genes and neurons mediate olfaction in C. elegans.</a> Cell. 74 (3), 515-527 (1993).</li><li>Sambongi, Y., 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=Caenorhabditis+elegans+senses+protons+through+amphid+chemosensory+neurons:+Proton+signals+elicit+avoidance+behavior.">Caenorhabditis elegans senses protons through amphid chemosensory neurons: Proton signals elicit avoidance behavior.</a> Neuroreport. 11 (10), 2229-2232 (2000).</li><li>Brenner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+of+Caenorhabditis+elegans.">The genetics of Caenorhabditis elegans.</a> Genetics. 77 (1), 71-94 (1974).</li><li>Brockie, P. J., Madsen, D. M., Zheng, Y., Mellem, J., Maricq, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+glutamate+receptor+subunits+in+the+nervous+system+of+Caenorhabditis+elegans+and+their+regulation+by+the+homeodomain+protein+UNC-42.">Differential expression of glutamate receptor subunits in the nervous system of Caenorhabditis elegans and their regulation by the homeodomain protein UNC-42.</a> Journal of Neuroscience. 21 (5), 1510-1522 (2001).</li><li>Brockie, P. J., Mellem, J. E., Hills, T., Madsen, D. M., Maricq, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+C.+elegans+glutamate+receptor+subunit+NMR-1+is+required+for+slow+NMDA-activated+currents+that+regulate+reversal+frequency+during+locomotion.">The C. elegans glutamate receptor subunit NMR-1 is required for slow NMDA-activated currents that regulate reversal frequency during locomotion.</a> Neuron. 31 (4), 617-630 (2001).</li><li>Gustafsson, B., Wingstrom, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+mechanisms+underlying+long-term+potentiation.">Physiological mechanisms underlying long-term potentiation.</a> Trends in Neuroscience. 11 (4), 156-162 (1988).</li><li>Kauer, J. A., Malenka, R. C., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+persistent+postsynaptic+modification+mediates+long-term+potentiation+in+the+hippocampus.">A persistent postsynaptic modification mediates long-term potentiation in the hippocampus.</a> Neuron. 1 (10), 911-917 (1988).</li><li>Bliss, T. V. P., Collingridge, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+synaptic+model+of+memory:+Long-term+potentiation+in+the+hippocampus.">A synaptic model of memory: Long-term potentiation in the hippocampus.</a> Nature. 361 (6407), 31-39 (1993).</li><li>Bailey, C. H., Giustetto, M., Huang, Y. Y., Hawkins, R. D., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+heterosynaptic+modulation+essential+for+stabilizing+Hebbian+plasticity+and+memory.">Is heterosynaptic modulation essential for stabilizing Hebbian plasticity and memory.</a> Nature Reviews Neuroscience. 1 (1), 11-20 (2000).</li><li>Miyashita, T., 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=Mg2++block+of+Drosophila+NMDA+receptors+is+required+for+long-term+memory+formation+and+CREB-dependent+gene+expression.">Mg2+ block of Drosophila NMDA receptors is required for long-term memory formation and CREB-dependent gene expression.</a> Neuron. 74 (5), 887-898 (2012).</li></ol>

The authors declare no conflicts of interest.

In the present study, all the reagents were kept at an RT of ~18 &#176;C on average, and animals were cultivated on a bench at the RT to avoid stress to the animals. Furthermore, all the experimental procedures were carried out at the RT. Animals were initially cultivated in an incubator at 20 &#176;C and then conditioned on a bench at ~24 &#176;C using reagents at the RT. Under these conditions, the outcomes of the conditioning were very variable. At the low RT, C. elegans grows slowly and should be cultivated longer than at 20 &#176;C until the animals reach the mature adulthood stage, as younger adult animals are more sensitive to the chemicals used for conditioning than mature adult animals and may show lower C.I. values.
The most critical step for successful conditioning is the washing of animals with ddH2O immediately after each chemical treatment. Therefore, mechanical and temperature stresses should be minimized by using sawed-off pipet tips, keeping reagents at RT, and very gently washing the animals by very slowly moving the animal collector up and down in ddH2O. Thorough washing of the animals each time after conditioning may affect learning and memory. The conditions of the chemotaxis assay plates also severely affect the outcomes. Too dry or too wet plates prevent smooth locomotion of the animals. Plates were prepared as described in step 1.; a good plate is one for which the 4 &#181;L spots of ddH2O or 5% aqueous 1-propanol are completely absorbed by the agar in approximately 5 min after spotting. As described above, animal ages are also critical for successful conditioning. Young adult animals are sensitive to mechanical and chemical treatment, resulting in variable outcomes, although very aged animals may not be suitable for conditioning either.
The shelf life of 1-propanol depends on brands and lots and is less than 3 months at RT. When the C.I. values of na&#239;ve animals get worse, it would be recommended to use fresh 1-propanol for the conditioning and chemotaxis assay.
The formation of memory by massed training was not affected by the treatment of animals with translation inhibitors (cycloheximide and anisomycin) and a transcription inhibitor (actinomycin D), while the formation of memory by the spaced training was markedly inhibited by the inhibitors20,21. Furthermore, the former memory decayed by cold shock, while the latter was retained for a longer period than the former and was resistant to cold shock. These results demonstrate that the former is STM and the latter is LTM, respectively20,21. However, the memory formed by the massed training may consist of STM and middle-term (intermediate-term) memory since STM is weakly dependent upon the CREB transcription factor (Figure 5A). This is consistent with the result that the STM was retained for more than 1 h20,21. The formation of both STM and LTM is highly dependent on nmr-1, which is expressed only in six pairs of neurons (AVA, AVD, AVE, RIM, AVG, and PVC) in C. elegans27,28. In these neurons, therefore, NMDA receptors may act as a molecular coincidence detector of 1% aqueous 1-propanol and HCl (pH 4.0) signals for synaptic plasticity, where the synaptic strengthening required for both STM and LTM can result from coincidental firing of pre- and post-synaptic neurons29,30,31,32,33.&#160;Therefore, the aversive associative memory may form among the interneurons.
The methods described in the present study should be applicable for appetitive olfactory learning and short-term and long-term associative memory using 1-nonanol as the CS and potassium chloride as the US21. It is interesting to compare the neuronal circuits that are involved in the formation of appetitive and aversive memories.

Learning and memory are vital for animals to survive and reproduce by efficiently navigating changing environments. C. elegans is an attractive model organism to study learning and memory at the molecular and cellular levels because of the simplicity of its nervous system, whose chemical and electrical wiring diagrams were completely reconstructed from serial electron micrographs of thin sections1,2,3.
C. elegans learns to associate cultivation temperature with starvation and migrates away from its growth temperature with an aversive memory lasting for several hours4,5.&#160;Conditioning C. elegans with sodium chloride (NaCl) in the absence of food leads to a reduction in chemotaxis toward NaCl6,7,8. When paired with food, butanone attraction is enhanced as a result of appetitive learning9,10,11. Although these phenomena are interpreted as associative learning and memory10,12, the distinction between associative learning and non-associative sensitization, habituation, and adaptation is not clear in the C. elegans learning and memory paradigm13,14. Indeed, animals conditioned with butanone and food deprivation (aversive conditioning) showed depressed coupling of the butanone sensory neuron AWCON to target neurons by insulin signals from other neurons, including AIA interneurons, while animals conditioned with butanone and food (appetitive conditioning) showed enhanced coupling of AWCON to target neurons15. The insulin signaling causes gene expression changes induced by nuclear EGL-4 and other transcriptional regulators16,17. Thus, this aversive and appetitive learning and memory has analogies with non-associative habituation and sensitization, respectively, of presynaptic sensory neurons in the gill-withdrawal reflex in Aplysia18,19.
By pairing two chemicals as the conditioned stimulus (CS) and unconditioned stimulus (US), we and others have developed protocols for the conditioning of C. elegans to form associative learning and memory without using food or starvation as the US20,21,22,23. In the present study, the protocols are modified to condition animals with 1-propanol and hydrochloric acid (HCl, pH 4.0) as the CS and US, respectively, for aversive learning and short-term memory (STM) and long-term memory (LTM). Na&#239;ve C. elegans is attracted by 1-propanol24 and repelled by acid25. When conditioned with a mixture of 1-propanol and HCl (pH 4.0), C. elegans was no longer or very weakly attracted to 1-propanol.

<table><tbody><tr><td>500 mL beaker</td><td>HARIO</td><td>B-500-H32</td><td></td></tr><tr><td>10 &amp;micro;L pipette tips</td><td>Thermo Fisher Scientific</td><td>H-104-96RS-Q</td><td></td></tr><tr><td>0.2 mL pipette tips</td><td>Thermo Fisher Scientific</td><td>TTW110RS-Q</td><td></td></tr><tr><td>1.0 mL pipette tips</td><td>Thermo Fisher Scientific</td><td>H-111-R100NS-Q</td><td></td></tr><tr><td>1.5 mL plastic tubes</td><td>Eppendorf</td><td>0030120086</td><td></td></tr><tr><td>2 mL plastic tubes</td><td>Eppendorf</td><td>0030120094</td><td></td></tr><tr><td>10 mL Serological pipettes</td><td>As One</td><td>2-5237-04</td><td></td></tr><tr><td>50 mL Serological pipettes</td><td>As One</td><td>2-5237-06</td><td></td></tr><tr><td>6-well cell culture plate</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>Aron Alpha (Glue for plastic)</td><td>Toagosei</td><td>High Speed EX</td><td></td></tr><tr><td>Autoclave</td><td>Tomy Digital Biology</td><td>SX-300</td><td></td></tr><tr><td>Bacto agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>Bacto peptone</td><td>BD</td><td>211677</td><td></td></tr><tr><td>Bottle top 0.2 &amp;micro;m filter units</td><td>Thermo Fisher Scientific</td><td>566-0020</td><td></td></tr><tr><td>Bunsen burner</td><td>EISCO</td><td>SKU&amp;nbsp;CH0089A</td><td></td></tr><tr><td>Calcium chloride dihydrate</td><td>Nacalai Tesque</td><td>06730-15</td><td></td></tr><tr><td>&lt;em&gt;C. elegans&lt;/em&gt; mutant strains</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>Cholesterol</td><td>Wako Pure Chemical Industries</td><td>034-03002</td><td></td></tr><tr><td>Clear acrylic cylindrical pipe</td><td>Asahi Kasei</td><td></td><td>3.5 cm (length), 30 mm (external diameter), 2 mm (thickness)</td></tr><tr><td>Crystallizing dish</td><td>Pyrex</td><td>3140-80</td><td></td></tr><tr><td>Dental burner</td><td>Phoenix-Dent</td><td>APT-3</td><td></td></tr><tr><td>Di-potassium hydrogen phosphate</td><td>Nacalai Tesque</td><td>28726-05</td><td></td></tr><tr><td>&lt;em&gt;E. coli &lt;/em&gt;OP50</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr><tr><td>Electric pipetter</td><td>Drummond Scientific</td><td>4-000-101</td><td></td></tr><tr><td>Gelatin</td><td>Wako Pure Chemical Industries</td><td>073-06295</td><td></td></tr><tr><td>Glass Petri dishes (10 cm in diameter)</td><td>As One&amp;nbsp;</td><td>Trade FLAT Mark</td><td></td></tr><tr><td>Heating magnetic stirrer</td><td>Thermo Fisher Scientific</td><td>SP131324</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Nacalai Tesque</td><td>37345-15</td><td></td></tr><tr><td>Incubator</td><td>SANYO</td><td>MIR-553</td><td></td></tr><tr><td>Kimwipes S-200</td><td>Nippon Paper Crecia</td><td>62011</td><td></td></tr><tr><td>Laboratory coat</td><td>TOYO LINT FREE</td><td>FH240C</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Nacalai Tesque</td><td>21002-85</td><td></td></tr><tr><td>Magnetic stirrer bar</td><td>SANSYO</td><td>93-5412</td><td></td></tr><tr><td>Metal spatula</td><td>FUJIFILM Wako</td><td>647-06531</td><td></td></tr><tr><td>Nitrile gloves</td><td>Kimberly-Clark</td><td>KC100</td><td></td></tr><tr><td>Nylon mesh (mesh size: 30 &amp;mu;m)</td><td>SEFAR</td><td>NY30-HD</td><td></td></tr><tr><td>P10 pipetman</td><td>Gilson</td><td>F144802</td><td></td></tr><tr><td>P200 pipetman</td><td>Gilson</td><td>F123600</td><td></td></tr><tr><td>P1000 pipetman</td><td>Gilson</td><td>F123602</td><td></td></tr><tr><td>pH meter</td><td>HORIBA&amp;nbsp;</td><td>Navi F-52</td><td></td></tr><tr><td>Plastic Petri dishes (9 cm in diameter)</td><td>IWAKI</td><td>SH90-15E</td><td></td></tr><tr><td>Plastic Petri dishes (6 cm in diameter)</td><td>SARSTEDT</td><td>82.1194.500</td><td></td></tr><tr><td>Plastic weighing boats</td><td>As One</td><td>1-5233-01</td><td></td></tr><tr><td>Platinum wire for a worm pick</td><td>Nilaco</td><td>PT-351265</td><td></td></tr><tr><td>1-Propanol</td><td>SIGMA-ALDRICH</td><td>279544</td><td></td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Nacalai Tesque</td><td>28721-55</td><td></td></tr><tr><td>Safety goggles</td><td>Kimberly-Clark</td><td>#25646</td><td></td></tr><tr><td>Sodium chloride</td><td>Nacalai Tesque</td><td>31320-05</td><td></td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Tooth picks</td><td></td><td></td><td></td></tr><tr><td>Water purification sysytem</td><td>Merck</td><td>Elix Essential 10 UV</td><td></td></tr><tr><td>Water urification sysytem</td><td>Merck</td><td>Milli-Q Synthesis A10</td><td></td></tr><tr><td>Weighing balance</td><td>METTLER&amp;nbsp;</td><td>TOREDO</td><td></td></tr><tr><td>Wild type &lt;em&gt;C. elegans&lt;/em&gt; strain N2</td><td>Caenorhabditis Genetics Center</td><td></td><td></td></tr></tbody></table>

aversive associative learning and memory formation by pairing two chemicals in caenorhabditis elegans

The nematode Caenorhabditis elegans is an attractive model organism to study learning and memory at molecular and cellular levels because of the simplicity of its nervous system, whose chemical and electrical wiring diagrams were completely reconstructed from serial electron micrographs of thin sections. Here, we describe detailed protocols for the conditioning of C. elegans by massed and spaced training for the formation of short-term memory (STM) and long-term memory (LTM), respectively. By pairing 1-propanol and hydrochloric acid as conditioned and unconditioned stimuli, respectively, C. elegans was successfully trained to form aversive associative STM and LTM. While na&#239;ve animals were attracted to 1-propanol, the trained animals were no longer or very weakly attracted to 1-propanol. Like in other organisms such as Aplysia and Drosophila, "learning and memory genes" play essential roles in memory formation. Particularly, NMDA-type glutamate receptors, expressed in only six pairs of interneurons in C. elegans, are required for the formation of both STM and LTM, possibly as a coincidence factor. Therefore, the memory trace may reside among the interneurons.

C. elegans was conditioned by massed training to form short-term aversive associative memory by pairing 1% aqueous 1-propanol and HCl (pH 4.0) as the CS and US, respectively. According to the protocol described above, synchronized animals were cultivated on a bench at an RT of 18 &#176;C for 5 days and were very gently washed 2x with ddH2O at an RT of 18 &#176;C. Then, the animals were conditioned with a mixture of 1% aqueous 1-propanol and HCl (pH 4.0) for 1 s. We also trained animals with ddH2O only, 1% aqueous 1-propanol only, and HCl (pH 4.0) only as references. After the conditioning, the animals were washed 1x with ddH2O. We repeated the conditioning 10x without interruption (no ITIs). Successful conditioning was achieved by repeating the procedure more than 7x up to 10x. Conditioning more than 10x resulted in less efficient learning21. After the training, the animals rested on bacterial food for 10 min at RT (18 &#176;C). After being washed with ddH2O 3x, the animals were transferred to a microcentrifuge tube by suspending in 0.25% aqueous gelatin and settled down to the bottom by gravity. After removing the supernatant as much as possible, the animals were gently resuspended in chemotaxis assay buffer and then allowed to settle down to the bottom of the tube by gravity.
After removing as much supernatant as possible, the animal suspension was spotted on the center circle of a chemotaxis assay plate, which was kept at an RT of 18 &#176;C, and then animals were allowed to freely move on the plate for 10 min at an RT of 18 &#176;C. C.I. values were calculated using the equation shown in Figure 3B. As shown in Figure 4A, animals conditioned with the mixture of 1% 1-propanol and HCl were no longer attracted to 5% 1-propanol spotted on agar plates for chemotaxis assay, whereas na&#239;ve and reference animals were similarly attracted to 5% 1-propanol. After the massed training (step 3.), the memory was no longer observed within 3 h20. Furthermore, the memory formed by the massed training was sensitive to cold shock20. These results demonstrate that C. elegans successfully formed aversive STM by massed training.
Animals were also conditioned by spaced training 10x with a 10 min ITI between the training steps (step 4.). During the ITI, the collector with animals was placed on a bacterial lawn on a 6 cm NGM plate at an RT of 18 &#176;C. Animals conditioned by the spaced training with a mixture of 1% aqueous 1-propanol and HCl (pH 4.0) were no longer attracted to 5% 1-propanol in comparison to animals treated with 1% 1-propanol only, HCl (pH 4.0) only, or ddH2O only (Figure 4B). After the spaced training, the animals retained the memory for more than 12 h20,21. Moreover, the memory did not form when animals were treated with translation or transcription inhibitors and was resistant to cold shock20,21. Therefore, C. elegans successfully formed aversive LTM by spaced training.
We also examined the effects of mutations in &#34;learning and memory genes&#34; on the formation of STM and LTM. The crh-1 gene encodes the ubiquitous transcription factor cAMP-response element-binding protein (CREB), glr-1 and nmr-1 encode &#945;-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA)-type and N-methyl-D-aspartate (NMDA)-type glutamate receptor subunits, respectively, and stau-1 encodes the double-stranded RNA-binding protein Staufen isoform. These genes play essential roles in classical conditioning in C. elegans, Drosophila, Aplysia, and mice. Using a mixture of 1% aqueous 1-propanol and HCl (pH 4.0), the formation of STM and LTM was dependent on all the genes (Figures 5A,B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64137/64137fig01.jpg" /> 
Figure 1: Experimental schematic of massed training. <a href="https://www.jove.com/files/ftp_upload/64137/64137fig01large.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/64137/64137fig02.jpg" /> 
Figure 2: Experimental schematic of spaced training. <a href="https://www.jove.com/files/ftp_upload/64137/64137fig02large.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/64137/64137fig03.jpg" /> 
Figure 3: Chemotaxis assay and chemotaxis index. (A) Schematic representation of a chemotaxis assay plate. Petri dishes (6 cm in diameter) were separated into four areas as shown, and 4 &#956;L each of 5% aqueous 1-propanol or ddH2O were diagonally spotted at two places each, 2 cm away from the center. (B) Chemotaxis index values were calculated from the equation shown by counting the number of animals in areas &#34;a&#34; and &#34;b&#34; after the completion of the chemotaxis. <a href="https://www.jove.com/files/ftp_upload/64137/64137fig03large.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/64137/64137fig04.jpg" /> 
Figure 4: Chemotaxis index values of animals conditioned with chemicals. Synchronized wild-type N2 animals were conditioned with chemicals indicated by (A) massed training 10x or (B) spaced training 10x. Flowcharts of the massed and spaced training protocols used are shown in Figure 1 and Figure 2, respectively. After the conditioning, the animals were free to move for 10 min on a 6 cm agar plate for chemotaxis assay at an RT of 18 &#176;C. C.I. values were calculated using the equation shown in Figure 3B. Data for this figure are provided in Supplemental Table 1. Data from the na&#239;ve animals were replotted in both figure panels. Bar plot shows the 1st quartile, median, and 3rd quartile. Asterisks (*P &#60; 0.05) indicate statistically significant differences determined by one-way ANOVA followed by Dunnett&#39;s multiple comparison test. <a href="https://www.jove.com/files/ftp_upload/64137/64137fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64137/64137fig05.jpg" /> 
Figure 5: Learning index values of conditioned mutant animals. Synchronized wild-type N2 and mutant animals indicated were conditioned with a mixture of 1% aqueous 1-propanol and HCl (pH 4.0) by (A) massed training 10x or (B) spaced training 10x. Flowcharts of the massed and spaced training protocols used are shown in Figure 1 and Figure 2, respectively. After the conditioning, the animals were free to move for 10 min on a 6 cm agar plate for chemotaxis assay at an RT of 18 &#176;C. Data for this figure are provided in Supplemental Table 2. Bar plot shows the 1st quartile, median, and 3rd quartile. Asterisks (*P &#60; 0.05) indicate statistically significant differences determined by one-way ANOVA followed by Dunnett&#39;s multiple comparison test. <a href="https://www.jove.com/files/ftp_upload/64137/64137fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: Young adult animals are sensitive to chemical treatment. Day 4 and day 5 wild-type N2 animals after hatching were massed-trained 10x with HCl, pH 4.0, without interruption and were then assayed for chemotaxis to 5% aqueous 1-propanol. Bars are means &#177; S.E.M. (n = 19). Asterisks (*P &#60; 0.05) indicate statistically significant differences determined by two-way ANOVA followed by the Tukey-Kramer post-hoc test. <a href="https://www.jove.com/files/ftp_upload/64137/Supplementary Figure S1.zip" target="_blank">Please click here to download this File.</a>
Supplemental Table 1: Data corresponding to Figure 4. <a href="https://www.jove.com/files/ftp_upload/64137/Supplemental Table S1.zip" target="_blank">Please click here to download this Table.</a>
Supplemental Table 2: Data corresponding to Figure 5. <a href="https://www.jove.com/files/ftp_upload/64137/Supplemental Table S2.zip" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans at JoVE.com

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

We have previously developed protocols for Caenorhabditis elegans to form short- and long-term associative memories by massed and spaced training, respectively. Here, detailed protocols are described for the conditioning of C. elegans by pairing 1-propanol and hydrochloric acid as conditioned and unconditioned stimuli, respectively, to form aversive associative memory.

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an attractive model organism to study learning and memory at molecular and cellular levels because of the ...

aversive-associative-learning-memory-formation-pairing-two-chemicals

Research

JoVE Journal

Neuroscience

2.3K Views.  Okinawa Institute of Science and Technology Graduate University. We have previously developed protocols for Caenorhabditis elegans to form short- and long-term associative memories by massed and spaced training, respectively. Here, detailed protocols are described for the conditioning of C. elegans by pairing 1-propanol and hydrochloric acid as conditioned and unconditioned stimuli, respectively, to form aversive associative memory.

Video: Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment through neuron-specific detection of food and chemical odors1, 2, and can associate nutritive states with chemical odors3, temperature4, and the pathogenicity of a food source5.
Here, we describe assays of C. elegans associative learning and short- and long-term associative memory. We modified an aversive olfactory learning paradigm6 to instead produce a positive response; the assay involves starving ~400 worms, then feeding the worms in the presence of the AWC neuron-sensed volatile chemoattractant butanone at a concentration that elicits a low chemotactic index (similar to Toroyama et al.7). A standard population chemotaxis assay1 tests the worms' attraction to the odorant immediately or minutes to hours after conditioning.
After conditioning, wild-type animals' chemotaxis to butanone increases ~0.6 Chemotaxis Index units, its "Learning Index". Associative learning is dependent on the presence of both food and butanone during training. Pairing food and butanone for a single conditioning period ("massed training") produces short-term associative memory that lasts ~2 hours. Multiple conditioning periods with rest periods between ("spaced training") yields long-term associative memory (&lt;40 hours), and is dependent on the cAMP Response Element Binding protein (CREB),6 a transcription factor required for long-term memory across species.8
Our protocol also includes image analysis methods for quick and accurate determination of chemotaxis indices. High-contrast images of animals on chemotaxis assay plates are captured and analyzed by worm counting software in MatLab. The software corrects for uneven background using a morphological tophat transformation.9 Otsu's method is then used to determine a threshold to separate worms from the background.10 Very small particles are removed automatically and larger non-worm regions (plate edges or agar punches) are removed by manual selection. The software then estimates the size of single worm by ignoring regions that are above a specified maximum size and taking the median size of the remaining regions. The number of worms is then estimated by dividing the total area identified as occupied by worms by the estimated size of a single worm.
We have found that learning and short- and long-term memory can be distinguished, and that these processes share similar key molecules with higher organisms.6,8 Our assays can quickly test novel candidate genes or molecules that affect learning and short- or long-term memory in C. elegans that are relevant across species.

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Here we describe methods to test C. elegans associative learning and short- and long-term associative memory. These population assays employ the worms abilities to chemotax toward volatile odorants, and form positive associations upon pairing food with the chemoattractant butanone. Increasing the number of conditioning periods induces long-term memory.

The memory of experiences and learned information is critical for organisms to make choices that aid their survival. C. elegans navigates its environment ...

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection of the neural circuits underlying these behaviors, the development of robust and reproducible behavioral assays is necessary. Previous C. elegans behavioral studies have used variations of a "drop test", a "chemotaxis assay", and a "retention assay" to investigate the response of C. elegans to soluble compounds. The method described in this article seeks to combine the complementary strengths of the three aforementioned assays. Briefly, a small circle in the middle of each assay plate is divided into four quadrants with the control and experimental solutions alternately placed. After the addition of the worms, the assay plates are loaded into a behavior chamber where microscope cameras record the worms' encounters with the treated regions. Automated video analysis is then performed and a preference index (PI) value for each video is generated. The video acquisition and automated analysis features of this method minimizes the experimenter's involvement and any associated errors. Furthermore, minute amounts of the experimental compound are used per assay and the behavior chamber's multi-camera setup increases experimental throughput. This method is particularly useful for conducting behavioral screens of genetic mutants and novel chemical compounds. However, this method is not appropriate for studying stimulus gradient navigation due to the close proximity of the control and experimental solution regions. It should also not be used when only a small population of worms is available. While suitable for assaying responses only to soluble compounds in its current form, this method can be easily modified to accommodate multimodal sensory interaction and optogenetic studies. This method can also be adapted to assay the chemosensory responses of other nematode species.

We present a behavior recording setup and protocol that enables automated analysis of the nematode, Caenorhabditis elegans' preference for soluble compounds in a population-based assay. This article describes the construction of a behavior chamber, the behavioral assay protocol, and video analysis software usage.

The nematode, Caenorhabditis elegans' compact nervous system of only 302 neurons underlies a diverse repertoire of behaviors. To facilitate the dissection ...

Behavior

Visual Classical Conditioning in Wood Ants

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, studies implementing classical conditioning paradigms with harnessed insects have been important for investigating the exact cues that individuals learn and the neural mechanisms underlying learning and memory formation. Here we present a protocol for evoking visual associative learning in wood ants through classical conditioning. In this paradigm, ants are harnessed and presented with a visual cue (a blue cardboard), the conditional stimulus (CS), paired with an appetitive sugar reward, the unconditional stimulus (US). Ants perform a Maxilla-Labium Extension Reflex (MaLER), the unconditional response (UR), which can be used as a readout for learning. Training consists of 10 trials, separated by a 5-minute intertrial interval (ITI). Ants are also tested for memory retention 10 minutes or 1 hour after training. This protocol has the potential to allow researchers to analyze, in a precise and controlled manner, the details of visual memory formation and the neural basis of learning and memory formation in wood ants.

We present a protocol for the classical conditioning of harnessed ants that permits researchers to study visual learning and memory formation at a level of analysis not possible with freely moving individuals.

Several species of insects have become model systems for studying learning and memory formation. Although many studies focus on freely moving animals, ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to passive avoidance behavior in laboratory rodents in which animals learn to avoid a compartment where they have previously received an electric shock. The assay takes advantage of negative geotaxis in flies, which manifests as an urge to climb up when they are placed on a vertical surface. The setup consists of vertically oriented upper and lower compartments. On the first trial, a fly is placed into a lower compartment from where it usually exits&#160;within 3-15 s, and steps into the upper compartment where it receives an electric shock. During the second trial, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first trial, indicating that flies formed long-term memory about the upper compartment. The recordings of latencies and number of shocks could be performed with a tally counter and a stopwatch or with an Arduino-based simple device. To illustrate how the assay can be used, the passive avoidance behavior of D. melanogaster and D. simulans male and female were characterized here. Comparison of latencies and number of shocks revealed that both D. melanogaster and D. simulans flies efficiently learned the passive avoidance behavior. No statistical differences were observed between male and female flies. However, males were a little faster while entering the upper compartment on the first trial, while females received a slightly higher number of shocks in every retention trial. The Western diet (WD) significantly impaired learning and memory in male flies while flight exercise counterbalanced this effect. Taken together, the passive avoidance behavior in flies offers a simple and reproducible assay that could be used for studying basic mechanisms of learning and memory.

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This work describes a simple behavioral paradigm that allows the analysis of aversive associative learning in adult fruit flies. The method is based on suppressing the innate negative geotaxis behavior due to the association formed between a specific environmental context and an electric shock.

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to ...

Extraction and Quantification of Volatile Sex Pheromone Responses in C. elegans

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and computations governing these signals remains an active area of research. The nematode Caenorhabditis elegans provides a powerful model for studying these processes as it produces a volatile sex pheromone. This pheromone is synthesized by virgin females or sperm-depleted hermaphrodites and serves as an attractant for males.
This protocol describes a detailed method for isolating the volatile sex pheromone from several C. elegans strains (WT strain N2, daf-22, and fog-2) and C. remanei. We also provide a protocol for quantifying the male chemotaxis response to the volatile sex pheromone. Our analysis utilizes measurements such as chemotaxis index (C.I.), arrival time (A.T.), and a trajectory plot to compare male responses under various conditions accurately. This method allows for robust comparisons between males of different genetic backgrounds or developmental stages. Furthermore, the experimental setup outlined here is adaptable to investigating other chemoattraction chemicals.

Author Spotlight: Examining Volatile Sex Pheromone Influence on Male C. elegans Behavior

This protocol establishes methods for extracting and quantifying responses to the volatile sex pheromone in C. elegans, providing tools to study chemical communication and navigation trajectory.

Chemical communication is vital in organismal health, reproduction, and overall well-being. Understanding the molecular pathways, neural processes, and ...

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 ...

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. Learning-induced changes in synaptic transmission are often distributed across many neurons and levels of processing in the brain. Therefore, methods to visualize learning-dependent synaptic plasticity across neurons are needed. The fruit fly Drosophila melanogaster represents a particularly favorable model organism to study neuronal circuits underlying learning. The protocol presented here demonstrates a way in which the processes underlying the formation of associative olfactory memories, i.e., synaptic activity and their changes, can be monitored in vivo. Using the broad array of genetic tools available in Drosophila, it is possible to specifically express genetically encoded calcium indicators in determined cell populations and even single cells. By fixing a fly in place, and opening the head capsule, it is possible to visualize calcium dynamics in these cells whilst delivering olfactory stimuli. Additionally, we demonstrate a set-up in which the fly can be subjected, simultaneously, to electric shocks to the body. This provides a system in which flies can undergo classical olfactory conditioning - whereby a previously na&#239;ve odor is learned to be associated with electric shock punishment - at the same time as the representation of this odor (and other untrained odors) is observed in the brain via two-photon microscopy. Our lab has previously reported the generation of synaptically localized calcium sensors, which enables one to confine the fluorescent calcium signals to pre- or postsynaptic compartments. Two-photon microscopy provides a way to spatially resolve fine structures. We exemplify this by focusing on neurons integrating information from the mushroom body, a higher-order center of the insect brain. Overall, this protocol provides a method to examine the synaptic connections between neurons whose activity is modulated as a result of olfactory learning.

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

Here we present a protocol with which pre- and/or postsynaptic calcium can be visualized in the context of Drosophila learning and memory. In vivo calcium imaging using synaptically localized calcium sensors is combined with a classical olfactory conditioning paradigm such that the synaptic plasticity underlying this type of associative learning may be determined.

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

C. elegans Chemotaxis Assay

Chemotaxis is a process in which cells or organisms move in response to a chemical stimulus. In nature, chemotaxis is important for organisms to sense and move toward food sources and move away from stimuli that may be toxic or harmful. Chemotaxis is also important at the cellular level. For example, chemotaxis is required for the movement of sperm toward an egg prior to fertilization. In the lab, chemotaxis is frequently examined in the nematode, C. elegans, which is known to migrate towards food sources in soil, but away from toxins such as heavy metals, substances with a low pH, and detergents. This video demonstrates how to perform a chemotaxis assay, which includes preparing the chemotaxis plates and the worms, running the assay, and analyzing the data. Then, we discuss examples of how chemotaxis assays can be used in C. elegans as a tool to understand learning and memory, olfactory adaptation, and neurological disease such as Alzheimer&quot;s disease. Chemotaxis experiments in C. elegans have near-limitless possibilities for learning more about the cellular and genetic mechanisms of many biological processes, and may lead to a greater understanding of human biology, development, and disease.

Chemotaxis Assay in Caenorhabditis elegans

Chemotaxis is a process in which cells or organisms move in response to a chemical stimulus. In nature, chemotaxis is important for organisms to sense and ...

Aversive Associative Learning and Memory Formation by Pairing Two Chemicals in Caenorhabditis elegans

Podsumowanie

Przeglądaj więcej filmów

Rozdziały w tym wideo

C. elegans Positive Butanone Learning, Short-term, and Long-term Associative Memory Assays

Automated Analysis of a Nematode Population-based Chemosensory Preference Assay

Visual Classical Conditioning in Wood Ants

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

Volatile Sex Pheromone Extraction and Chemoattraction Assay in Caenorhabditis elegans

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay

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

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

C. elegans Chemotaxis Assay