Volume 91

2013

An NRC Research Press Journal

Une revue deNRC Research Press

Avec le concours de la Société canadienne de zoologie

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Revue canadienne de

zoologie

Canadian Journal of

Zoology

In cooperation with the Canadian Society of Zoologists

REVIEW

From likes to dislikes: conditioned taste aversion in the great pondsnail (Lymnaea stagnalis)1

E. Ito, S. Kojima, K. Lukowiak, and M. Sakakibara

Abstract: The neural circuitry comprising the central pattern generator (CPG) that drives feeding behavior in the great pondsnail (Lymnaea stagnalis (L., 1758)) has been worked out. Because the feeding behavior undergoes associative learning andlong-termmemory (LTM) formation, it provides an excellent opportunity to study the causal neuronal mechanisms of these twoprocesses. In this review, we explore some of the possible causal neuronal mechanisms of associative learning of conditionedtaste aversion (CTA) and its subsequent consolidation processes into LTM in L. stagnalis. In the CTA training procedure, a sucrosesolution, which evokes a feeding response, is used as the conditioned stimulus (CS) and a potassium chloride solution, whichcauses a withdrawal response, is used as the unconditioned stimulus (US). The pairing of the CS–US alters both the feedingresponse of the snail and the function of a pair of higher order interneurons in the cerebral ganglia. Following the acquisitionof CTA, the polysynaptic inhibitory synaptic input from the higher order interneurons onto the feeding CPG neurons isenhanced, resulting in suppression of the feeding response. These changes in synaptic efficacy are thought to constitute a“memory trace” for CTA in L. stagnalis.

Key words: conditioned taste aversion, feeding, long-term memory, Lymnaea stagnalis, withdrawal.

Résumé : Les circuits neuronaux constituant le générateur central de patrons (CPG) qui régit le comportement d’alimentationde la grande limnée (Lymnaea stagnalis (L., 1758)) ont été décrits. Étant donné que le comportement d’alimentation est assujetti al’apprentissage associatif et a la formation de la mémoire a long terme (LTM), il présente une excellente occasion d’étudier lesmécanismes neuronaux causaux de ces deux processus. Dans la présente synthèse, nous examinons certains de mécanismesneuronaux causaux possibles de l’apprentissage associatif de l’aversion gustative conditionnée (CTA) et les processus de consol-idation subséquente associés dans la LTM de L. stagnalis. Dans la procédure d’apprentissage de la CTA, une solution de sucrose,qui provoque une réaction d’alimentation, est utilisée comme stimulus conditionné (CS), et une solution de chlorure depotassium, qui provoque une réaction de retrait, est utilisée comme stimulus non conditionné (US). Le jumelage de CS–USmodifie la réaction d’alimentation de l’escargot et la fonction d’une paire d’interneurones d’ordre supérieur dans le ganglioncérébral. Après l’acquisition de la CTA, le signal synaptique inhibiteur polysynaptique des interneurones d’ordre supérieur versles neurones associés a l’alimentation du CPG est rehaussé, entraînant la suppression de la réaction d’alimentation. Ceschangements de l’efficacité synaptique constitueraient une « trace de la mémoire » pour la CTA chez les L. stagnalis. [Traduit parla Rédaction]

Mots-clés : aversion gustative conditionnée, alimentation, mémoire a long terme, Lymnaea stagnalis, retrait.

IntroductionInmany respects, the birth ofmodern neuroscience occurred in

the 1950s. In our view, two seminal events happened. The first wasthe brain surgery performed on a patient known as HM that leadsto Milner’s observations of human memory which ultimatelyshowed that hippocampal neural circuits were necessary for theformation of declarative memory (Milner et al. 1998). Those obser-vations ultimately lead to the proliferation of studies concernedwith the neuronal changes that occurred within the hippocampalcircuits which are necessary for memory formation. However, thetechniques and knowledge needed to undertake those studiesdepended on a second happening, the realization that molluscspossess large, identifiable neurons which controlled interesting,tractable behaviors.

For example, early studies occurring about the same timein France and Monaco by Tauc (1954) and Arvanitaki andChalazonitis (1955) using the central nervous system (CNS) of the

sea hare (genus Aplysia L., 1758) laid the foundation for the use ofthese model systems to study the causal neuronal mechanisms oflearning and memory. We can get an appreciation of this situa-tion by reading the report of Strumwasser (1971). He wrote that “Ihad come to Woods Hole to receive instruction from AngeliqueArvanitaki and her husband Nick Chalazonitis in the methodol-ogy work on the Aplysia CNS. In 1955, Arvanitaki and Chalazonitisand quite independently, Tauc, had performed the first cellularrecordings from the large neurons of Aplysia.” We feel that Kandeland Tauc's (1965) discovery of heterosynaptic facilitation in a mol-luscan preparation laid the groundwork for hypotheses devel-oped later to explain the neuronal basis of learning and thesubsequent formation of memory. These studies utilizing for themost part molluscan preparations (California seahare, Aplysiacalifornica J.G. Cooper, 1863) culminated in Kandel being awardedthe Nobel Prize for Medicine and Physiology in 2000 “for the discover-ies concerning signal transduction in the nervous system” (Kandel2001).

Received 6 November 2012. Accepted 4 February 2013.

E. Ito. Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, 1314-1 Shido, Sanuki 769-2193, Japan.S. Kojima. Sandler Neurosciences Center, University of California, San Francisco, 675 Nelson Rising Lane 518, San Francisco, CA 94143-0444, USA.K. Lukowiak. Hotchkiss Brain Institute, University of Calgary, Calgary, AB T2N 4N1, Canada.M. Sakakibara. School of High-Technology for Human Welfare, Tokai University, 317 Nishino, Numazu 410-0321, Japan.

Corresponding author: Etsuro Ito (e-mail: eito@kph.bunri-u.ac.jp).1This review is one of a series dealing with trends in the biology of the phylum Mollusca.

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Can. J. Zool. 91: 405–412 (2013) dx.doi.org/10.1139/cjz-2012-0292 Published at www.nrcresearchpress.com/cjz on 8 March 2013.

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In addition to the genus Aplysia, many other gastropodmolluscswere used in these early days of neuroscience to perfect the intra-cellular techniques still used today in attempting to elucidate thecausalmechanisms ofmemory formation. Great strides have beenmade by a number of groups using such model systems. Forexample, Alkon and his colleagues have used the marine snailhermissenda (Hermissenda crassicornis (Eschscholtz, 1831)). Theyconcentrated their efforts on associative learning by developing aclassical conditioning procedure utilizing light as the conditionedstimulus (CS) and vibration as the unconditioned stimulus (US)(Alkon 1975). Their data showed that a specific type of photorecep-tor, the B-type, was a key site for long-term memory (LTM) forma-tion (Ito et al. 1994; Kawai et al. 2004a). Crow continues to usePavlovian conditioning of H. crassicornis (Crow and Tian 2006).Gelperin and his group demonstrated the remarkable learningand memory capabilities of the giant garden slug (Limax maximusL., 1758) (Gelperin 1975). Matsuo and his colleagues are enthusias-tically advancing the cellular and molecular neurobiology of thethree-band garden slug (Limax valentianus (Férussac, 1823)) (Matsuoet al. 2011).

The great pond snail (Lymnaea stagnalis (L., 1758)) (Fig. 1) is an-other useful gastropod mollusc and has become an importantmodel system for studying the causal neuronal mechanisms ofassociative learning and the subsequent formation of LTM. Theinitial studies utilizing L. stagnalis to study the associative learninginvolved in feeding behaviors were begun in the 1980s (Alexanderet al. 1982; Audesirk et al. 1982; Kemenes and Benjamin 1989a,1989b), and some of the studies in the subsequent decades utilizedboth classical and operant conditioning of a number of differentbehaviors, including various aspects of feeding,withdrawal, and aer-ial respiratory behaviors (Kemenes and Benjamin 1994; Lukowiaket al. 1996; Whelan and McCrohan 1996; Kemenes et al. 1997, 2011;Hermann and Bulloch 1998; Staras et al. 1998a, 1998b; Spencer et al.1999; Kawai et al. 2004b; Straub et al. 2004; Sakakibara 2006; Suzukiet al. 2008; Kita et al. 2011). In addition, CNS preparations have also

been utilized to study neural analogues of associative learning invitro (Veprintsev and Rozanov 1967; Kemenes et al. 1997; Sunadaet al. 2012).

In our opinion, the most important reason for adapting theL. stagnalis model system to study learning and memory is the factthat the underlying neuronal circuitry has beenworked out betterthan in any other model system to study associative learning. Webase this opinion on the following facts. (1) The underlying neu-ronal circuitry of the central pattern generator (CPG) that drivesfeeding behavior has been worked out better than in other mol-luscan preparations (Benjamin and Rose 1979; Rose and Benjamin1979; McCrohan and Benjamin 1980; Elliott and Benjamin 1985a,1985b; Benjamin et al. 2000, 2008; Benjamin 2012). (2) The CPGthat drives aerial respiration is the only neuronal circuit that weknow of where both the sufficiency and the necessity of the3-neuron circuit has been experimentally demonstrated; in addi-tion, one of the three CPG neurons, RPeD1, has been shown to bea necessary site for LTM formation (Syed et al. 1990, 1992;Lukowiak 1991; Winlow and Syed 1992; Taylor and Lukowiak 2000;Scheibenstock et al. 2002; Sangha et al. 2003a, 2003b; Lukowiaket al. 2010). (3) Lymnaea stagnalis feeding behaviors undergo bothappetitive and aversive classical conditioning (Whelan andMcCrohan 1996; Ito et al. 1999; Staras et al. 1999a, 1999b; Kawai et al.2004b; Straub et al. 2006). (4) Aerial respiratory behavior can beoperantly conditioned, and the memory formed following learningcanbemodifiedbyenvironmentally relevant stimuli (Lukowiaket al.1996, 1998, 2000, 2003a, 2003b, 2003c, 2008, 2010). (5) Both feedingand aerial respiration are tractable behaviors that exhibit associativelearning (classical conditioning and operant conditioning) and thesubsequent consolidationof the learning intoLTM (Azamiet al. 2006;Fulton et al. 2008; Teskey et al. 2012). (6) Finally, both behaviorsundergoone-trial learning that leads toLTMformation,whichallowsinvestigators to more accurately investigate the time course of themolecular and neural events leading to LTM formation (Alexanderet al. 1984; Fulton et al. 2005; Martens et al. 2007; Sugai et al. 2007).

Fig. 1. The great pond snail (Lymnaea stagnalis). All the snails used were originally the gift of Vrije Universiteit Amsterdam and have beenmaintained in our laboratories. We generally used snails with a 20 mm shell length.

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Thus, because the neuronal circuitry has been so well worked outand the behaviors mediated by those circuits exhibit associativelearning and LTM formation, the advantages offered by the L. stagna-lismodel systemare second to none. This point has been brought outpreviously by Chase (2002) in his excellent book comparing variousmolluscan preparations.

In the present review, we present an outline of cellular mecha-nisms underlying aversive conditioning in the feeding behavior ofL. stagnalis.

Conditioned taste aversion in L. stagnalisThe Ito group, with help from M. Sakakibara and K. Lukowiak,

has so far noted one remarkable learning ability in L. stagnalis. Thisis the capacity to establish taste aversion and consolidate it intoLTM. This phenomenon is referred to as conditioned taste aver-sion (CTA) (Kojima et al. 1996). To produce CTA in L. stagnalis, anappetitive stimulus (e.g., sucrose) is used as the CS. Application ofthe CS to the lips increases the feeding response (i.e., the numberof bites) in snails. An aversive stimulus (e.g., KCl) is used as the US.Application of the US to the snails inhibits feeding behavior. Inthe taste aversion training procedure, the CS is pairedwith the US.After repeated temporal contingent presentations of the CS andUS, the CS no longer elicits a feeding response (Fig. 2), and thistaste aversion persists for more than a month (Kojima et al. 1996).

Enhancement of the inhibition on feeding centralpattern generator neurons

Based on the above behavioral experiments, we proposed aworking hypothesis for CTA in L. stagnalis (Figs. 3A, 3B). We hy-pothesized that when the CS (sucrose) is followed by the US (KCl)in the training session, the association of the CS and US causes apotentiation of an inhibitory neuronal pathway, resulting in sup-

pression of the feeding response to the CS (Fig. 3A; Kojima et al.1996). Taking into account the underlying neural circuits workedout by the Benjamin group (Benjamin and Elliott 1989; Fergusonand Benjamin 1991a, 1991b; Syed and Winlow 1991; Elliott andKemenes 1992; McCrohan and Kyriakides 1992; Inoue et al. 1996a,1996b; Yeoman et al. 1994a, 1994b, 1996; Staras et al. 1998b;Kemenes et al. 2001; Straub and Benjamin 2001; Straub et al. 2002),our model further proposes that sensory neuron(s) activated bythe appetitive sucrose (CS) excite the feeding CPG neurons whichdrive motor neurons in the CS pathway to induce a feeding re-sponse. Similarly, sensory neuron(s) activated by the aversive KClstimulus (US) excite withdrawal interneurons that activate motorneurons in the US pathway, resulting in a withdrawal response.The withdrawal response takes precedence over the feeding re-sponse. With the pairing of CS–US, the CS is no longer capable ofeliciting feeding. It is the association of the CS and US in the keyinterneurons that result in the CS no longer being able (i.e., whileLTM persists) to elicit the feeding response (Fig. 3B).

Previous studies have shown that the cerebral giant cells (CGCs)exert both aweak excitatorymonosynaptic influence and a stronginhibitory polysynaptic influence on the neuron 1 medial (N1Mcells) of the feeding CPG, and that the repetitive firing of the CGCsresults in inhibitory influences on the N1M cells (Yeoman et al.1996). The CGCs act as a pair of interneurons, one located in theright and the other in the left cerebral ganglia. We showed thatthe CS and US are associated in the CGCs and alter the activity ofthe CGCs (Nakamura et al. 1999a, 1999b). Because we applied CSand US only to the lips, but not the neurons in the CNS, thesesolutions were used as tastes for the lips, such as sweet and bitter,respectively. The concentrations of the sucrose solution (CS) andthe KCl solution (US) that we used in our conditioning paradigmare each 10mmol/L.We have in control experiments shown that ifthese solutions are directly applied to the CNS, no responses arerecorded at the CGCs. Thus, the CGCs were a logical site for fur-ther explanation to elucidate the neuronal mechanisms of CTA.

With further experiments, we found that a polysynaptic inhib-itory postsynaptic potential (IPSP) recorded in the N1M cells byactivation of the CGCs was larger and lasted longer in the tasteaversion trained snails than that in the control snails (Fig. 4;Kojima et al. 1997). These data suggested to us that an enhancedIPSP in the N1M cells underlies the suppression of feeding re-sponse in the CTA of L. stagnalis. Interestingly, when the amplitudeof the IPSP recorded in the N1M cells in the CNS taken from goodmemory performers was compared with the IPSP amplitude re-corded in the poor memory performers, we found that there wasa much greater variance in the amplitude of the IPSP from thepoor performers. This suggested to us that this greater variance inIPSP amplitude in the poor performers corresponds to the instabilityof the input elicited by the US in those key target neurons. Thus, thepolysynaptic IPSP from the CGCs to the N1M cells in memory-poorperformers is not able to suppress the feeding response.

Multiple site optical analysis of conditioned tasteaversion

When we published our electrophysiological data and our in-terpretation of those data, we received a substantial amountof criticism. The criticism primarily centered on whether thechanges we observed (i.e., the long-lasting synaptic change be-tween the CGCs and the N1M cells) were the only changes thatoccurred in the CNS of the taste aversion trained snails. In otherwords, we had no information about any other changes occurringin synaptic strength in other CNS neurons. To attempt to answerthis criticism, we used an optical recording technique to measurechanges that could occur in other CNS neurons in response totaste aversion training (Kojima et al. 2001). To perform these ex-periments, we used isolated CNS preparations obtained from tasteaversion trained snails, stained them with a voltage-sensitive dye

Fig. 2. Learning scores after taste aversion training in the greatpond snail (Lymnaea stagnalis). Taste aversion training was broughtabout by pairing 10 mmol/L sucrose (conditioned stimulus: CS) and10 mmol/L KCl (unconditioned stimulus: US). The duration of boththe CS and the US was 15 s, with an interstimulus interval betweenthe onsets of CS and US of 15 s. A 10 min intertrial interval wasinterposed between each pairing of the CS–US. Snails received10 paired CS–US trials. We also used a backward conditioned (US–CS)control group and a naive control group to validate associativelearning. For the naive control group, only distilled water wasapplied to the lips instead of the CS and US. The y axis shows thenumber of bites/min after training that was evoked by applicationof sucrose (i.e., CS). The learning score by taste aversion training(CTA) is better when snails are trained in the morning than in theafternoon (see Wagatsuma et al. 2004). Data are expressed as themean + SE. Figure appears in colour on the Journal’s Web site.

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RH155, and simulated the presentation of sucrose (i.e., the CS) withelectrical stimulation of the median lip nerve. Themedian lip nervetransmits chemosensory signals of appetitive taste to the CNS.

We optically detected a large number of spikes in several areasof the buccal ganglion after electrical stimulation of the medianlip nerve. The effects of behavioral taste aversion training on thespike responses were examined in two areas of the buccal gan-glion where the most active neural responses were seen. In onearea that accounted for the N1M cells, the number of spikes aftermedian lip nerve stimulation (i.e., the simulated CS) was signifi-cantly reduced in taste aversion trained snails compared wih con-trol snails. In another area positioned between the buccal motorneurons (i.e., the B3 motor neuron and the B4 cluster cells), theevoked spike responses elicited by median nerve stimulationwere unaffected in the taste aversion trained preparations. Thesedata showed that the appetitive signal transmitted via themedianlip nerve to the N1M cells is suppressed following CTA. This resultsin a decrease of the fictive feeding response. However, even withour optical recording technique, we still cannot rule out the pos-sibility that changes in neuronal activity in other areas of the CNSoccur with taste aversion training.

Memory trace in the feeding central patterngenerator in conditioned taste aversion

As described above, the polysynaptic IPSP recorded in the N1Mcells by activation of the CGCs in taste aversion trained snails waslarger and lasted longer than the IPSP in control snails (Fig. 4).However, the neural circuit between the CGC and the N1M cellconsists of two types of synaptic connections: (1) the excitatorymonosynaptic connection from the CGC to the neuron 3 tonic

Fig. 3. Our working hypothesis for conditioned taste aversion in the great pond snail (Lymnaea stagnalis). (A) Neuromodulatory model. Whensucrose (CS) is followed by KCl (US) in the training session, the association of these stimuli occurs at one or more loci in the central nervoussystem. Then this association enhances an inhibitory pathway, resulting in suppression of the feeding response to sucrose (CS). (B) Neuralcircuitry model. The sensory neurons (SNs) sensitive to sucrose excite the interneurons (INs), including the feeding central pattern generatorneurons, and the motor neurons (MNs) to induce a feeding response, whereas the sensory neurons to KCl excite the interneurons and themotor neurons in the withdrawal pathway, resulting in a withdrawal response. Based on previous observations by many researchers, wehypothesized that a pair of cerebral giant cells (CGCs) receive the information of the above two stimuli and that the CGC exerts a strongpolysynaptic inhibitory influence on one of the feeding central pattern generator neurons (neuron 1 medial (N1M) cell) via the neuron 3 tonic(N3t) cell. Modified from Kojima et al. (1997). Figure appears in colour on the Journal’s Web site.

Fig. 4. Enhancement of polysynaptic inhibitory postsynapticpotential (IPSP) in the neuron 1 medial (N1M) cells by activation ofthe cerebral giant cells (CGCs) after taste aversion training in thegreat pond snail (Lymnaea stagnalis). The CGCs were depolarized for2 s. The IPSP was larger and lasted longer (two-way repeated-measuresANOVA, P < 0.01) in the taste aversion trained snails (CTA) than inthe naive or backward snails (control). *, P < 0.05; **, P < 0.01. Dataare expressed as the mean ± SE. Modified from Kojima et al. (1997).Figure appears in colour on the Journal’s Web site.

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(N3t) cell and (2) the inhibitory monosynaptic connection fromthe N3t cell to the N1M cell (Fig. 5A). As a next step, we had todetermine which synaptic connection is more changed followingthe acquisition of CTA.

The recent studies on appetitive conditioning of feeding behav-ior in L. stagnalis by the Benjamin group made three points (Marraet al. 2010). (1) Tonic inhibition in the feeding network is providedby the N3t cell. This interneuron makes a monosynaptic inhibi-tory connection with the N1M cell. (2) There is a reduction in N3tspiking after appetitive conditioning, and this reduction in N3tfiring inversely correlates with an increase in the conditionedfictive feeding response. (3) Computer simulation of N3t–N1M in-teractions suggests that changes in N3t firing are sufficient toexplain the increase in the fictive feeding activity produced byappetitive conditioning. These data showed that appetitive condi-tioning of feeding behavior in L. stagnalis occurs because of thecombined effects of reduced tonic inhibition and enhanced excit-

atory synaptic connections between the CS pathway and the feed-ing command neurons (Fig. 5A).

Next, we hypothesized that “taste aversion learning” would oc-cur via a mechanism which was the inverse of the mechanismproposed for “appetitive conditioning”. That is, there would be anincrease in N3t spiking after conditioning, and this increase inN3t firing would inversely correlate with a reduction in the con-ditioned fictive feeding response. We thus hypothesized thattaste aversion learning in L. stagnalis is also due to the combinedeffects of reduced tonic inhibition and enhanced excitatorysynaptic connections between the CS pathway and the feedingcommand neurons.

However, because the N3t cells are too small to access consis-tently by standard sharp electrode recording techniques, in thepresent study the synaptic inputs from the CGCs to the N3t cellsand those from the N3t cells to the N1M cells were inferred bymonitoring the monosynaptic excitatory postsynaptic potential(EPSP) recorded in the large B1 and B3 motor neurons, respec-tively. The evokedmonosynaptic EPSPs of the B1motor neurons inthe brains isolated from the taste aversion trained snails wereidentical to those in the control snails, whereas the spontaneousmonosynaptic EPSPs recorded in the B3 motor neurons were sig-nificantly enlarged (Fig. 5B; Ito et al. 2012).

These data suggested that, after taste aversion training, themonosynaptic inputs from the N3t cells to the follower neurons,including the N1M cells, are facilitated. That is, one of the neuralcorrelates of CTA–LTM is an increase in neurotransmitter releasefrom the N3t cells. We thus conclude that the N3t cells suppressthe N1M cells in the feeding CPG, in response to the CS inL. stagnalis CTA.

Development of the life cycle and development ofthe learning ability

By using the taste aversion training procedures in L. stagnalis, wecan assess the common changes that occur both development ofthe life cycle and development of the learning ability of this or-ganism (Ono et al. 2002; Karasawa et al. 2008; Sunada et al. 2010a).We examined developmental changes in the acquisition and re-tention of CTA in L. stagnalis (Yamanaka et al. 1999). Our datashowed that snails developed their ability to form CTA–LTMthrough the three critical stages: (1) stage 25 embryos (veliconcha)start to respond to appetitive sucrose, (2) stage 29 embryos justbefore hatching acquire CTA, but not LTM, and (3) immature snailswith a 10mm shell are able to learn and remember which foods canbe safely eaten. That is, the development of learning ability in snailsis coincident with the major changes in their life cycle.

We then examined the relationship between the learning abil-ity for CTA and the development of the CGC for CTA. UsingLucifer-yellow staining of the CGCs and Azan staining for theganglion sections, we found that the CGCs mature at the earlydevelopmental stages and that the number of buccal and cerebralneurons in immature snails is similar to that seen in adult snails(Sadamoto et al. 2000). The immunoreactivity of serotonin, whichis one of the main neurotransmitters employed in the feedingcircuitry (Kemenes et al. 1989, 1997; Kemenes 1997; Hatakeyamaand Ito 1999; Nakamura et al. 1999c; Kawai et al. 2011), was firstobserved in the CGCs at stage 29 (Yamanaka et al. 2000). Afterhatching, the neuropile of CGCs developed faster than other cellsin the buccal and cerebral ganglia, resulting in their early inner-vation at the immature stage. Thus, the developmental changes inthe CGCs correlate well with the ability to form CTA.

Identification of interneurons involved in thewithdrawal response that affect the cerebral giantcells

Although we have some evidence that there are input pathwaysonto the CGCs fromhigher order interneurons whichmediate the

Fig. 5. Enhancement of spontaneous excitatory postsynapticpotential (EPSP) in the B3 motor neurons after taste aversiontraining in the great pond snail (Lymnaea stagnalis). (A) Schematicpresentation of the neural circuitry underlying taste aversiontraining. The signals of sucrose (conditioned stimulus: CS) and KCl(unconditioned stimulus: US) are associated in the cerebral giantcells (CGCs). Rectangles and circles indicate interneurons and motorneurons, respectively. At synapses, open circles and solid circlesindicate excitatory monosynaptic inputs and inhibitorymonosynaptic inputs, respectively. The neuron 1 medial (N1M),neuron 2 (N2), and neuron 3 tonic (N3t) cells form part of thefeeding central pattern generator (CPG). (B) Spontaneous EPSP in theB3 motor neurons. The EPSP recorded in the B3 motor neurons canbe used for monitoring the changes in the N3t–N1M synapticconnection. The B3 EPSPs recorded from the taste aversion trainedsnails were significantly larger (one-way ANOVA, P < 0.05) thanthose observed for the backward conditioned and naive controlsnails. Data are expressed as the mean + SE. Modified from Ito et al.(2012). Figure appears in colour on the Journal’s Web site.

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withdrawal response elicited by the KCl stimulus (Nakamura et al.1999a, 1999b), these interneurons have not been positively identi-fied (Ferguson and Benjamin 1991a, 1991b). However, two identi-fied neurons are good candidates. One is the pleural–buccalneuron (PlB) and the other is the right pedal dorsal 11 neuron(RPeD11). The PlB is FMRFamidergic andwas reported to inhibit allthe neurons in the feeding circuit, including protraction and re-traction motor neurons, feeding CPG interneurons, buccal modu-latory interneurons, and the CGCs (Alania et al. 2004, 2008).

On the other hand, the Sakakibara group with the help ofK. Lukowiak demonstrated that the RPeD11 sends an inhibitory in-put onto the CGCs (Sunada et al. 2012). The RPeD11, a well-knowninterneuron receiving sensory input from the right parietal dorsal3 (RPD3) and sending output to the motor cluster neurons rightpedal G (RPeG) and the right cerebral A (RCeA), exerts withdrawalbehavior in response to multimodal noxious stimuli such as me-chanical prodding, KCl application, and shadow presentation(Sunada et al. 2010b). An aversive stimulus to L. stagnalis employedin CTA as a US basically resulted in withdrawal behavior. TheSakakibara group’s studies on neuronal mechanisms in the CTAused sucrose application as the CS andweakmechanical proddingto the animal’s head as the US (Kawai et al. 2004b). After acquisi-tion of learning, the conditioned animals responded to decreasesin the feeding response against the CS application. The presenta-tion of the US, irrespective of whether KCl application or mechan-ical prodding was used, increased excitability in the RPeD11. Evenwith the application of weak mechanical prodding, the RPeD11 wasexcited, thereby decreasing the feeding response. The strong excita-tion inducedbypositive current injection into theRPeD11 resulted ininhibition of the CGCs, as evidenced by such effects as a decrease inspontaneous firing activity. This inhibitory effect was transmitted tothe CGCs via mono-chemical synapses. The isolated preparationswithmouth, buccal, and esophageal gangliamay provide a commonplatform for the CTA in vitro conditioning model using sucrose ap-plication as the CS and current injection into the RPeD11 as the US(Sunada et al. 2012).

Future questionsIn our studies designed to determine how long CTA–LTM per-

sists, it became apparent that snails continued to eat their normaldiet of lettuce or similar leafy plants in their home aquaria whilestill exhibiting CTA–LTM. Thus, it was unclear what the relation-ship was between a CTA for a specific CS and other appetitive foodstimuli. If snails can successfully differentiate between appetitivefood stimuli, where in the CNS does this occur? Our previousexperiments showed that snails can be differentially conditionedto avoid one appetitive CS following taste aversion training whilecontinuing to be responsive to a different appetitive food CS thathas not been paired in a forward manner with an aversive US(Sugai et al. 2006). That is, L. stagnalis can distinguish betweentastes during CTA. The neurons responsible for taste discrimina-tion may be located in the CNS and most probably exist upstreamof the CGCs, but we have to carefully address this question andattempt to find neurons involved in taste discrimination in theL. stagnalis CNS in the future.

ConclusionResearchers investigating CTA in rats and other mammals are

often surprised to see that relatively simple invertebrate modelsystems, such as L. stagnalis, are also capable of acquiring CTA. Infact, one author (Bernstein 1999) concluded from our data that theneural circuitry required for this learning is fairly primitive. How-ever, we consider that this perceived weakness of the L. stagnalismodel is actually an advantage (e.g., Murakami et al. 2013), be-cause the use of simple invertebrate systems can provide answersto important basic questions before wemove on tomore complexmammalian systems.

The molecular mechanisms that regulate serotonin release fromtheCGCshave also been clarified in L. stagnalis. Thekeyplayers in themolecular cascades are cAMP, protein kinase A, cAMP response ele-ment binding protein, CCAAT/enhancer binding protein, and sero-tonin transporter (Nakamura et al. 1999c; Sadamoto et al. 2004a,2004b, 2008, 2010, 2011; Hatakeyama et al. 2004a, 2004b, 2006;Wagatsuma et al. 2005, 2006). We think that regulation of theamount of serotonin released from the CGCs plays an importantrole in CTA. These cascades will be reviewed elsewhere.

AcknowledgementsThis work was supported by KAKENHI from the Japan Society

for the Promotion of Science (JSPS No. 21657022) to E.I. and grantsfrom the Canadian Institutes of Health Research (CIHR) and theNatural Sciences and Engineering Research Council of Canada(NSERC) to K.L.

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