Several gastropod molluscs ( How-To- Do- It and slugs) have been shown capable of classical conditioning. Recently the freshwater stagnalis (figure 1) has joined the list of gas- Classical in tropods shown capable of being con- Conditioning the PondSnail ditioned (Audesirk, Alexander, Au- desirk, & Moyer 1982; Alexander, Lymnaeastagnalis Audesirk, & Audesirk 1982, 1984). The method used was nonaversive classical conditioning of feeding, in JamesE. which a novel, nonfood chem- Alexander,Jr. ostimulus (amyl acetate, CS) was TeresaE. Audesirk paired with food (sucrose and casein GeraldJ. Audesirk digest, UCS). The snails, after several CS-UCS pairings, subsequently re- sponded to the CS as food. This pa- , in coping with the daily responds to a formerly inadequate per describes in detail a technique for problems encountered in an ever- stimulus (CS) that has been associ- classically conditioning Lymnaeathat changing environment, must show ated with an adequate stimulus (UCS) can be conducted by high school or Downloaded from http://online.ucpress.edu/abt/article-pdf/47/5/295/9842/4448054.pdf by guest on 30 September 2021 considerable malleability in their be- was eventually called classical condi- college students in an introductory havior. Evolution has shaped mem- tioning. psychology or behavior labo- bers of a particularspecies to be more Since the publication of Pavlov's pi- ratory course. The experimental pro- or less alike in morphology, phys- oneering work, classical conditioning cedure is flexible, rapid, simple, and iology, and behavior, all of which are and other forms of learning have inexpensive. under substantial genetic control. Ex- been demonstrated in many animals tensive as it is, though, the genetic in a wide variety of conditions. A tre- blueprint of an animal cannot provide mendous body of data exists in the Materials specific answers to all of the daily psychological literature concerning For raising and housing Lymnaea: problems individual animals face conditioning under controlled condi- throughout their lives. Instead, the tions in several animals such as rats 1. (1) 10 gal aquarium, with filtering genome codes for the development of and pigeons. Learningalso occurs un- apparatus a nervous system, with its attendant der a variety of natural conditions, 2. (3) 5 gal plastic containers for properties of change and storage, particularly when the animal's sur- holding water which allows the animal to learn and vival and/or successful reproduction 3. Piece of chalk or small amount of to be capable of altering its activity on are at stake. A good example of the calcium carbonate the basis of past experience. adaptive value of conditioning is the 4. TetraMin flaked fish food or let- The experimental procedure de- association between an ingested food tuce scribed in this paper, classical condi- and illness in blue jays. A blue jay, 5. Small amount of fine-grained tioning, was developed by the noted getting sick after eating a poisonous sand Russian physiologist Ivan Pavlov. butterfly, learns to associate the ill- 6. (1) indelible marker, black (for After a neutral, ineffective stimulus (a ness with the consumption of the but- marking snails) bell) which he called the conditioned terfly and subsequently avoids similar 7. (1) tube of Super Glue (for mark- stimulus (CS), Pavlov presented meat butterflies (Brower 1969). The learn- ing snails) powder to dogs (the unconditioned ing of birdsongs, imprinting in pre- For training Lymnaea(per student stimulus, or UCS) which elicited a sal- cocial birds, and the memorization of or group of students): ivation response from the dogs. After the location of flowers and their hives repeated paired presentations of the by bees are other examples of learn- 1. (6) plastic bins, 15 x 22 x 5 cm two stimuli: the bell (CS) and meat ing in the wild. (Rubbermaid) powder (UCS), Pavlov discovered that the bell presented alone became capable of eliciting salivation. This learning paradigm, where the animal

James Alexander has a B.A. and an M.A. from the Univ. of Missouri. His master's thesis was on learningin gastropodmol- luscs.He is now finishinghis Ph.D. at the Univ. of Oklahoma.Teresa Audesirk and Gerald Audesirk share a position at the University of Colorado-Denver. Teresa is investigating the neuronal basis of chem- oreceptionin the gastropod,Lymnaea. She holds a B.A. from Bucknell Univ. and a Ph.D. from USC. Gerald is conducting re- A B C search on the effects of chronic low-level lead exposure on individual neurons. He FIGURE1. A. Lymnaeastagnalis. B. This animal is about to rasp (comparewith C). C. This earned his B.S. from Rutgers and his snail is in the middle of a rasp, the behavioral response to be observed. The mouth has Ph.D. from Caltech. opened, and the radulais about to extend through the mouth.

CLASSICALCONDITIONING IN POND SNAILS 295 2. (2) pieces of plastic or fiberglass around 20 to 25? C. Feces should be ment. Lymnaeacan live for more than screening, cut, molded, and sewn removed and some of the water in the a week without food. to fit the inner dimensions of the tank replaced periodically to remove One last point-gently handle the above bins wastes. The snails can be fed flaked snails throughout testing and training 3. (1) piece of screening, 2 x 3 ft fish food (TetraMin) or lettuce ad Ii- in order to minimize disturbance. 4. CS training stock solution (240 ul bitum.TetraMin results. in much more (microliter)amyl acetate added to rapid growth. Day 1 11 of aerated tap water) 5. UCS training stock solution (40 g Experimental Procedure Assign Snails to Treatment sucrose added to 1 1 aerated tap Groups. water) An importantpoint to be stressed is 6. (7) plastic measuring cups, 100 ml the flexibilityof the experimental pro- In order to compare the responses capacity cedure. For purposes of description of individual snails, each snail should 7. (1) timer, 1-6 min and discussion, it is assumed that the be individually numbered. Remove 20 8. (1) 5 gal plastic container students will have two hours a day large (about 0.5 g, although larger or 9. 5 1 of aerated tap water (per day) for five days to conduct the experi- smaller animals can be used) snails ment. The five days do not need to be from the tank per student, place the For testing Lymnaea(per student or consecutive. The total number of snails on a wet piece of paper towel, group of students): training trials is six, three trials a day foot downward, and allow the snails'

for two days. Students can work in shells to dry (about 15 minutes). Downloaded from http://online.ucpress.edu/abt/article-pdf/47/5/295/9842/4448054.pdf by guest on 30 September 2021 1. (10) glass beakers, 120-150 ml ca- small groups instead of individually. Clearly mark a different identifying pacity Hunger motivation is important. number on the largest part of the 2. (1) ring stand, 8-9 inches tall, 4-5 Hunger affects performance, as seen shell (the body whorl) of each snail inches in diameter in Audesirk et al. (1982). The snails with a black indelible marker. After 3. (1) piece of plate glass, 5 x 5 should be starved for two days prior allowing the marks to dry (about 10 inches to the start and during the experi- minutes), apply a thin coating of Su- 4. (1) large round mirror, 4-5 inches in diameter 5. (3) pocket mirrors,2 x 3 inches 6. (2) plastic syringes, 6 ml capacity 7. (2) plastic measuring cups, 100 ml capacity PLASTICSCREENING 8. CS testing stock solution (400 ul (microliter)of amyl acetate added to 1 1 of aerated tap water) 9. 5 1 of aerated tap water (per day) 10. (1) timer, 1-6 min dollar/ 11. (1) counter (grocery story HOME BIN cent counter or lab hand-held counter) Lymnaeacan be raised easily in the laboratory from eggs or young ani- mals. Lymnaeais a freshwater RINSEBIN HOMEBIN found in lakes, streams, and ponds throughout the northern latitudes of North America and Europe. An aquarium filled with aerated tap SOLUTIONS water is adequate for raising Lymnaea, but several precautions should be A noted. Copper is very toxic to snails; water from copper pipes could kill these animals. If the water supply is chlorinated, the water should be -R MIRRORS treated with chemicals or aerated to remove the chlorine. Aerating the SYRINGES PLATEGLASS water in several plastic 5 gallon con- tainers by bubbling air through the RING STAND water will remove the chlorine. Lymnaeathrives best in slightly al- kaline conditions. Additions of a small amount of chalk or calcium car- bonate will assure alkalinity. A small TIMER WATER D MIRROR amount of fine-grained sand also CS should be added because sand parti- SOLUTION BEAKERSWITH TEST ANIMALS cles are necessary for the trituration COUNTER of food. The tank should be situated B out of direct sunlight. Water tem- perature should be maintained FIGURE2. A. Trainingapparatus. B. Testing apparatus.

296 THE AMERICAN BIOLOGY TEACHER, VOLUME 47, NO. 5, MAY 1985 per Glue over the marks, being very careful not to get any of the glue on TABLE1. Testing Procedure the snails' bodies. After this coating feels completely dry (about 15 min- 1. snails placed in number-codedbeakers (40 ml water) utes), place 10 snails in each of two 2. snails acclimate for 15 minutes plastic bins (lined with the screening) 3. beaker placed on viewing stand containing 200 ml of aerated tap 4. 5 ml of water added to beaker via syringe water. Label one bin "Experimental 5. rasps counted for 2 minutes (pre-CScount) Group" and the other "Control 6. 5 ml of CS test stock solution added to beakervia syringe Group", and note which snails are in 7. rasps counted for 2-minute period (post-CS count) which group. Keep the bins covered 8. water in beaker poured off and fresh water added to beaker with the 2 x 3 ft piece of screening to 9. after all snails tested, place snails back into home bins prevent snails from escaping. Constructionof the viewing platform: A viewing platform is necessary for kept in a stoppered bottle, since observationof the movements and ac- vigorously just prior to use) via sy- best is somewhat volatile. tions of the snails during testing. ringe. Count the number of rasps amyl acetate The final UCS concentration that the Glue the to the of the made in the two-minute period fol- plate glass top is 0.67 percent-a ring stand (aquarium sealant or wax lowing the addition of the CS. After snails experience concentration sufficient enough to works well for this). Situate the large testing, pour off the water and re- round mirrorat a 45 degree angle be- place with fresh aerated water. Set cause rasping. Downloaded from http://online.ucpress.edu/abt/article-pdf/47/5/295/9842/4448054.pdf by guest on 30 September 2021 each student or group will low the top of the ring stand, and this snail aside and test the next snail. First, of six bins, two bins that place the three pocket mirrors, taped After all snails have been tested, need a total experimental animals (la- end to end, on top of the stand (fig- place them back into their appropriate house the beled appropriately), and four more ure 2B). home bins. Individual snail's scores are generated by subtracting the bins, filled with 200 ml of aerated tap spontaneous rasping activity (the pre- water (figure 2A). Label two of the Day 2 CS count) from that elicited by the CS four bins "Rinse", one bin "Experi- Group", and the last bin GatheringBaseline Data. (the post-CS count). If the CS is inhib- mental itory, the score for that particular "Control Group". Place one "Rinse" On the second day, test the animals snail could be negative, if the CS is bin between the two groups labeled to observe their naive reaction to the excitatory,the score will be positive. "ExperimentalGroup" (one of these CS (amyl acetate) and to establish a previously labeled and containingthe baseline for future performance (see experimental animals), and place the Table 1). Place each animal in a num- Days 3 and 4 other "Rinse" bin between the two bered glass beaker containing 40 ml of "Control Group" bins (one already aerated tap water, recording the Training Trials. containing the control animals). Label snail's number and the beaker Training occurs on the 3rd and 4th two of the measuring cups "CS", two number. After allowing the snails to days. The animals will receive a total cups "UCS", and the last three cups acclimate (at least 15 minutes), gently of six training trials over this two-day "H20". Place the timer nearby. place a beaker on the viewing plat- period, three trials a day with an in- Pour 50 ml of the CS training stock form, and record the beaker number. tertrialinterval (the time between suc- solution into a "CS" cup, and 50 ml Add 5 ml of aerated tap water (a dis- cessive training trials) of 30 minutes of the UCS into an "UCS" cup, and turbance control) slowly via syringe, (Table2). Each day, the experimental place these cups in front of the bin aiming the jet of water at the wall op- animals receive three CS-UCS pair- housing the experimental animals. posite the snail's position. Then count ings at 30-minute intervals, alternat- Pour 50 ml of the UCS into another each rasp (see below) made by the ing with three H20-H20 presenta- "UCS"cup, and 50 ml of aerated tap snail in the next two minutes. This tions. The control group receives water into an "H20" cup, and place generates a baseline of spontaneous separate, alternating presentations of these cups in front of the home bin rasping activity. the CS and UCS. Since the control housing the control animals. The The response to be measured is the group consequently receives more water serves as a substitute CS to number of stereotyped feeding move- handling, thus more disturbance, the equalize disturbancebetween the two ments, or rasps. A rasp consists of experimentalgroup receives the H20- groups. Slowly pour the CS into the the following sequence of events. H20 presentations in order to equal- bin housing the experimental ani- First, the mouth opens, and the ize the amount of disturbance be- mals, distributing the CS throughout radula, a rough tongue-like organ, ex- tween the two groups. The final CS the bin while avoiding pouring it di- tends through the mouth and scrapes concentration that the snails experi- rectly on the snails. Then pour the along the substrate in a posterior to ence is the same in training and test- water in the same manner into the anteriordirection. The radula then re- ing, 0.004 percent. The CS solution is bin containing the control animals. tracts into the mouth, the mouth closes, and the sequence repeats (see figure 1). Students should familiarize themselves with this response by pri- TABLE2. TrainingProcedure or observation of snails feeding in the tank. Time(min) ExperimentalGroup ControlGroup After counting the number of spon- taneous rasps, add 5 ml of the amyl 0,30,60 CS-UCS, rinse, transfer water-UCS,rinse, transfer acetate testing stock solution (the so- 15,45,75 water-water, rinse, transfer CS-water,rinse, transfer lution should be made and shaken

CLASSICAL CONDITIONING IN POND SNAILS 297 After this, pour the UCS into the ex- that affect learning. The following are can use the t-test for comparing the perimental animals' bin and the UCS a few suggestions for expanding the response of the experimental group to into the control animals' bin. Work at objectives of this experiment. that of the control group before and a pace so that the UCS is added to the Influence of food-deprivation:some after training. In addition, a paired t- experimentalbin roughly 15 seconds students can compare the response of test can be run to compare the experi- after the addition of the CS. Set the a group of well-fed snails (allowed to mental group's response after train- timer for two minutes and leave the feed on 1 g TetraMinfor five minutes ing to that observed before training. If experimental animals in the CS-UCS daily) to that of food-deprived snails snails are repeatedly tested, the re- mixture and the control animals in (snails that have been starved one sults could be analyzed using a three- the presence of the UCS for two min- week prior to and during the experi- way analysis of variance with repeat- utes. ment). Hunger influences leaming as ed testing. After two minutes, lift the screens seen in Audesirk et al. (1982). (and thus the snails) gently out of the Influence of the number of training References home bins (figure 2A) and place the trials:a group of students could com- screens briefly (about 15 seconds) into pare the response of snails given one Audesirk, T.E., Alexander, J.E. Jr., Au- the "Rinse"bins. Then lift the screens CS-UCSpresentation versus that of desirk, G.J., & Moyer, C. (1982). Rapid, out again and place the screens into snails trained with 10 or 20 training 'non-aversive conditioning in a fresh- the appropriate new home bins (fig- trials. water gastropod. I. Effects of age and motivation. Behavioraland NeuralBiology, ure 2A). Empty, rinse, replace, and Extinction:some students could ob- 36, 379-390. refill the rinse bins and old home bins serve the length of time required for Alexander, J.E. Jr., Audesirk, T.E., & Au- Downloaded from http://online.ucpress.edu/abt/article-pdf/47/5/295/9842/4448054.pdf by guest on 30 September 2021 with 200 ml of fresh aerated water in snails to 'forget' the learned response. desirk, G.J. (1982). Rapid, non-aversive preparationfor the next presentation. Also, these students could see if dif- conditioning in a freshwater gastropod. Fifteen minutes after the UCS presen- ferences in extinction rates occur be- II. Effects of temporal relationships on tation, pour 50 ml of the CS training tween groups of snails given different learning. Behavioraland Neural Biology, stock solution, followed 15 seconds numbers of training trials, or between 36, 391-402. laterby 50 ml of aerated water (a sub- groups of snails given the same Alexander, J.E. Jr., Audesirk, T.E., & Au- stitute for UCS) into the bin contain- number of training trials, but with the desirk, G.J. (1984). One-trial, long last- ing reward leaming in the snail Lymnaea ing the control snails. At the same trials presented over a different stagnalis.The Journal of Neurobiology,15, time, pour 50 ml of aerated water (a amount of time (see below). 67-72. substitute CS) followed 15 seconds Influence of time between training Brower, L. P. (1969). Ecologicalchemistry. later by 50 ml of water (a substitute trials: students could compare the ef- ScientificAmerican, 220, 22-29. UCS) in the bin housing the experi- fects of 10 training trials in one day mental group. Rinse and transfer versus 10 training trials spread out both groups two minutes later. Emp- over a five-day period. The time inter- ty, rinse, and refill the rinse and old val between trials affects conditioning home bins with 200 ml of fresh water. in Lymnaeaas seen in Alexander et al. Alternate between these two types (1982). of presentations until the experimen- Different control groups: different tal group has received three CS-UCS control groups could be used and presentations and the control group compared with each other and with receives three separate presentations the experimental group. Students of the CS and UCS. After this has thus can discover the importance of been accomplished, training is done control groups in learning. For fur- for the day. ther discussion of this topic, see Au- desirk et al. (1982). Manipulating the temporal rela- Day 5 tionship between the CS and UCS: some students can investigate this Post-trainingTesting. important parameter of learning, the On the fifth day of the experiment temporal relationship between the ...... test each snail again to observe the ef- two stimuli of classical conditioning. fect of trainingon the response to the They can compare the responses of CS. The testing procedure for this test snails trained under a forward delay (the post-training test) is exactly the conditioning procedure (where the same as the testing procedure prior to CS precedes and overlaps with the training (the pre-training test-see UCS) to snails trained under a back- Water IoiIand air. sampIing test Table 1). The snails should be placed ward delay conditioning procedure equime. ent design:ed and as-: in numbered beakers and the snail (the UCS precedes and overlaps with sembled for educational app.li and beaker numbers should be re- the CS). For further discussion see Al- cations. From Imnology to.vo.ag training, from elementary science corded by an assistant so that the stu- exander et al. (1982). programs to professional field dents doing the testing are unaware stud ies. F or c l assroom or fiel.d . of the groups to which the snail be- *tri:p.All ..the way.. itoi.-olleg.e.. ...e longs. StatisticalAnalysis After generating means and stand- Alternative Experiments ard deviations of rasps for experimen- tal and control groups before and Chestertown' Ma$rylad 21620 Some students may wish to investi- after training, students can plot the 301 778O-1 gate the influences of other variables results and analyze the data. Students

298 THEAMERICAN BIOLOGY TEACHER, VOLUME 47, NO. 5, MAY1985