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Canadian Journal of

Zoology

Volume 91

An NRC Research Press Journal

2013

Une revue de NRC Research Press

www.nrcresearchpress.com

Revue canadienne de

zoologie

In cooperation with the Canadian Society of Zoologists

Avec le concours de la Société canadienne de zoologie

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

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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 pond snail (Lymnaea stagnalis (L., 1758)) has been worked out. Because the feeding behavior undergoes associative learning and long-term memory (LTM) formation, it provides an excellent opportunity to study the causal neuronal mechanisms of these two processes. In this review, we explore some of the possible causal neuronal mechanisms of associative learning of conditioned taste aversion (CTA) and its subsequent consolidation processes into LTM in L. stagnalis. In the CTA training procedure, a sucrose solution, which evokes a feeding response, is used as the conditioned stimulus (CS) and a potassium chloride solution, which causes a withdrawal response, is used as the unconditioned stimulus (US). The pairing of the CS–US alters both the feeding response of the snail and the function of a pair of higher order interneurons in the cerebral ganglia. Following the acquisition of CTA, the polysynaptic inhibitory synaptic input from the higher order interneurons onto the feeding CPG neurons is enhanced, 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’alimentation de la grande limnée (Lymnaea stagnalis (L., 1758)) ont été décrits. Étant donné que le comportement d’alimentation est assujetti a` l’apprentissage associatif et a` la formation de la mémoire a` long terme (LTM), il présente une excellente occasion d’étudier les mécanismes neuronaux causaux de ces deux processus. Dans la présente synthèse, nous examinons certains de mécanismes neuronaux causaux possibles de l’apprentissage associatif de l’aversion gustative conditionnée (CTA) et les processus de consolidation 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 de potassium, qui provoque une réaction de retrait, est utilisée comme stimulus non conditionné (US). Le jumelage de CS–US modifie la réaction d’alimentation de l’escargot et la fonction d’une paire d’interneurones d’ordre supérieur dans le ganglion cérébral. Après l’acquisition de la CTA, le signal synaptique inhibiteur polysynaptique des interneurones d’ordre supérieur vers les neurones associés a` l’alimentation du CPG est rehaussé, entraînant la suppression de la réaction d’alimentation. Ces changements de l’efficacité synaptique constitueraient une « trace de la mémoire » pour la CTA chez les L. stagnalis. [Traduit par la Rédaction] Mots-clés : aversion gustative conditionnée, alimentation, mémoire a` long terme, Lymnaea stagnalis, retrait.

Introduction In many respects, the birth of modern neuroscience occurred in the 1950s. In our view, two seminal events happened. The first was the brain surgery performed on a patient known as HM that leads to Milner’s observations of human memory which ultimately showed that hippocampal neural circuits were necessary for the formation of declarative memory (Milner et al. 1998). Those observations ultimately lead to the proliferation of studies concerned with the neuronal changes that occurred within the hippocampal circuits which are necessary for memory formation. However, the techniques and knowledge needed to undertake those studies depended on a second happening, the realization that molluscs possess large, identifiable neurons which controlled interesting, tractable behaviors. For example, early studies occurring about the same time in France and Monaco by Tauc (1954) and Arvanitaki and Chalazonitis (1955) using the central nervous system (CNS) of the

sea hare (genus Aplysia L., 1758) laid the foundation for the use of these model systems to study the causal neuronal mechanisms of learning and memory. We can get an appreciation of this situation by reading the report of Strumwasser (1971). He wrote that “I had come to Woods Hole to receive instruction from Angelique Arvanitaki and her husband Nick Chalazonitis in the methodology work on the Aplysia CNS. In 1955, Arvanitaki and Chalazonitis and quite independently, Tauc, had performed the first cellular recordings from the large neurons of Aplysia.” We feel that Kandel and Tauc's (1965) discovery of heterosynaptic facilitation in a molluscan preparation laid the groundwork for hypotheses developed later to explain the neuronal basis of learning and the subsequent formation of memory. These studies utilizing for the most part molluscan preparations (California seahare, Aplysia californica J.G. Cooper, 1863) culminated in Kandel being awarded the Nobel Prize for Medicine and Physiology in 2000 “for the discoveries concerning signal transduction in the nervous system” (Kandel 2001).

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: [email protected]). 1This review is one of a series dealing with trends in the biology of the phylum Mollusca.

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

In addition to the genus Aplysia, many other gastropod molluscs were used in these early days of neuroscience to perfect the intracellular techniques still used today in attempting to elucidate the causal mechanisms of memory formation. Great strides have been made by a number of groups using such model systems. For example, Alkon and his colleagues have used the marine snail hermissenda (Hermissenda crassicornis (Eschscholtz, 1831)). They concentrated their efforts on associative learning by developing a classical conditioning procedure utilizing light as the conditioned stimulus (CS) and vibration as the unconditioned stimulus (US) (Alkon 1975). Their data showed that a specific type of photoreceptor, the B-type, was a key site for long-term memory (LTM) formation (Ito et al. 1994; Kawai et al. 2004a). Crow continues to use Pavlovian conditioning of H. crassicornis (Crow and Tian 2006). Gelperin and his group demonstrated the remarkable learning and memory capabilities of the giant garden slug (Limax maximus L., 1758) (Gelperin 1975). Matsuo and his colleagues are enthusiastically advancing the cellular and molecular neurobiology of the three-band garden slug (Limax valentianus (Férussac, 1823)) (Matsuo et al. 2011). The great pond snail (Lymnaea stagnalis (L., 1758)) (Fig. 1) is another useful gastropod mollusc and has become an important model system for studying the causal neuronal mechanisms of associative learning and the subsequent formation of LTM. The initial studies utilizing L. stagnalis to study the associative learning involved in feeding behaviors were begun in the 1980s (Alexander et al. 1982; Audesirk et al. 1982; Kemenes and Benjamin 1989a, 1989b), and some of the studies in the subsequent decades utilized both classical and operant conditioning of a number of different behaviors, including various aspects of feeding, withdrawal, and aerial respiratory behaviors (Kemenes and Benjamin 1994; Lukowiak et 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; Suzuki et al. 2008; Kita et al. 2011). In addition, CNS preparations have also

been utilized to study neural analogues of associative learning in vitro (Veprintsev and Rozanov 1967; Kemenes et al. 1997; Sunada et al. 2012). In our opinion, the most important reason for adapting the L. stagnalis model system to study learning and memory is the fact that the underlying neuronal circuitry has been worked out better than in any other model system to study associative learning. We base this opinion on the following facts. (1) The underlying neuronal circuitry of the central pattern generator (CPG) that drives feeding behavior has been worked out better than in other molluscan preparations (Benjamin and Rose 1979; Rose and Benjamin 1979; McCrohan and Benjamin 1980; Elliott and Benjamin 1985a, 1985b; Benjamin et al. 2000, 2008; Benjamin 2012). (2) The CPG that drives aerial respiration is the only neuronal circuit that we know of where both the sufficiency and the necessity of the 3-neuron circuit has been experimentally demonstrated; in addition, one of the three CPG neurons, RPeD1, has been shown to be a 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; Lukowiak et al. 2010). (3) Lymnaea stagnalis feeding behaviors undergo both appetitive and aversive classical conditioning (Whelan and McCrohan 1996; Ito et al. 1999; Staras et al. 1999a, 1999b; Kawai et al. 2004b; Straub et al. 2006). (4) Aerial respiratory behavior can be operantly conditioned, and the memory formed following learning can be modified by environmentally relevant stimuli (Lukowiak et al. 1996, 1998, 2000, 2003a, 2003b, 2003c, 2008, 2010). (5) Both feeding and aerial respiration are tractable behaviors that exhibit associative learning (classical conditioning and operant conditioning) and the subsequent consolidation of the learning into LTM (Azami et al. 2006; Fulton et al. 2008; Teskey et al. 2012). (6) Finally, both behaviors undergo one-trial learning that leads to LTM formation, which allows investigators to more accurately investigate the time course of the molecular and neural events leading to LTM formation (Alexander et al. 1984; Fulton et al. 2005; Martens et al. 2007; Sugai et al. 2007). Published by NRC Research Press

Ito et al.

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

Thus, because the neuronal circuitry has been so well worked out and the behaviors mediated by those circuits exhibit associative learning and LTM formation, the advantages offered by the L. stagnalis model system are second to none. This point has been brought out previously by Chase (2002) in his excellent book comparing various molluscan preparations. In the present review, we present an outline of cellular mechanisms underlying aversive conditioning in the feeding behavior of L. stagnalis.

Conditioned taste aversion in L. stagnalis The Ito group, with help from M. Sakakibara and K. Lukowiak, has so far noted one remarkable learning ability in L. stagnalis. This is the capacity to establish taste aversion and consolidate it into LTM. This phenomenon is referred to as conditioned taste aversion (CTA) (Kojima et al. 1996). To produce CTA in L. stagnalis, an appetitive stimulus (e.g., sucrose) is used as the CS. Application of the CS to the lips increases the feeding response (i.e., the number of bites) in snails. An aversive stimulus (e.g., KCl) is used as the US. Application of the US to the snails inhibits feeding behavior. In the taste aversion training procedure, the CS is paired with the US. After repeated temporal contingent presentations of the CS and US, the CS no longer elicits a feeding response (Fig. 2), and this taste aversion persists for more than a month (Kojima et al. 1996).

Enhancement of the inhibition on feeding central pattern generator neurons Based on the above behavioral experiments, we proposed a working hypothesis for CTA in L. stagnalis (Figs. 3A, 3B). We hypothesized that when the CS (sucrose) is followed by the US (KCl) in the training session, the association of the CS and US causes a potentiation of an inhibitory neuronal pathway, resulting in sup-

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pression of the feeding response to the CS (Fig. 3A; Kojima et al. 1996). Taking into account the underlying neural circuits worked out by the Benjamin group (Benjamin and Elliott 1989; Ferguson and Benjamin 1991a, 1991b; Syed and Winlow 1991; Elliott and Kemenes 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 by the appetitive sucrose (CS) excite the feeding CPG neurons which drive motor neurons in the CS pathway to induce a feeding response. Similarly, sensory neuron(s) activated by the aversive KCl stimulus (US) excite withdrawal interneurons that activate motor neurons in the US pathway, resulting in a withdrawal response. The withdrawal response takes precedence over the feeding response. With the pairing of CS–US, the CS is no longer capable of eliciting feeding. It is the association of the CS and US in the key interneurons that result in the CS no longer being able (i.e., while LTM persists) to elicit the feeding response (Fig. 3B). Previous studies have shown that the cerebral giant cells (CGCs) exert both a weak excitatory monosynaptic influence and a strong inhibitory polysynaptic influence on the neuron 1 medial (N1M cells) of the feeding CPG, and that the repetitive firing of the CGCs results in inhibitory influences on the N1M cells (Yeoman et al. 1996). The CGCs act as a pair of interneurons, one located in the right and the other in the left cerebral ganglia. We showed that the CS and US are associated in the CGCs and alter the activity of the CGCs (Nakamura et al. 1999a, 1999b). Because we applied CS and US only to the lips, but not the neurons in the CNS, these solutions were used as tastes for the lips, such as sweet and bitter, respectively. The concentrations of the sucrose solution (CS) and the KCl solution (US) that we used in our conditioning paradigm are each 10 mmol/L. We have in control experiments shown that if these solutions are directly applied to the CNS, no responses are recorded at the CGCs. Thus, the CGCs were a logical site for further explanation to elucidate the neuronal mechanisms of CTA. With further experiments, we found that a polysynaptic inhibitory postsynaptic potential (IPSP) recorded in the N1M cells by activation of the CGCs was larger and lasted longer in the taste aversion trained snails than that in the control snails (Fig. 4; Kojima et al. 1997). These data suggested to us that an enhanced IPSP in the N1M cells underlies the suppression of feeding response in the CTA of L. stagnalis. Interestingly, when the amplitude of the IPSP recorded in the N1M cells in the CNS taken from good memory performers was compared with the IPSP amplitude recorded in the poor memory performers, we found that there was a much greater variance in the amplitude of the IPSP from the poor performers. This suggested to us that this greater variance in IPSP amplitude in the poor performers corresponds to the instability of the input elicited by the US in those key target neurons. Thus, the polysynaptic IPSP from the CGCs to the N1M cells in memory-poor performers is not able to suppress the feeding response.

Multiple site optical analysis of conditioned taste aversion When we published our electrophysiological data and our interpretation of those data, we received a substantial amount of criticism. The criticism primarily centered on whether the changes we observed (i.e., the long-lasting synaptic change between the CGCs and the N1M cells) were the only changes that occurred in the CNS of the taste aversion trained snails. In other words, we had no information about any other changes occurring in synaptic strength in other CNS neurons. To attempt to answer this criticism, we used an optical recording technique to measure changes that could occur in other CNS neurons in response to taste aversion training (Kojima et al. 2001). To perform these experiments, we used isolated CNS preparations obtained from taste aversion trained snails, stained them with a voltage-sensitive dye Published by NRC Research Press

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Fig. 3. Our working hypothesis for conditioned taste aversion in the great pond snail (Lymnaea stagnalis). (A) Neuromodulatory model. When sucrose (CS) is followed by KCl (US) in the training session, the association of these stimuli occurs at one or more loci in the central nervous system. Then this association enhances an inhibitory pathway, resulting in suppression of the feeding response to sucrose (CS). (B) Neural circuitry model. The sensory neurons (SNs) sensitive to sucrose excite the interneurons (INs), including the feeding central pattern generator neurons, and the motor neurons (MNs) to induce a feeding response, whereas the sensory neurons to KCl excite the interneurons and the motor neurons in the withdrawal pathway, resulting in a withdrawal response. Based on previous observations by many researchers, we hypothesized that a pair of cerebral giant cells (CGCs) receive the information of the above two stimuli and that the CGC exerts a strong polysynaptic 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 postsynaptic potential (IPSP) in the neuron 1 medial (N1M) cells by activation of the cerebral giant cells (CGCs) after taste aversion training in the great pond snail (Lymnaea stagnalis). The CGCs were depolarized for 2 s. The IPSP was larger and lasted longer (two-way repeated-measures ANOVA, P < 0.01) in the taste aversion trained snails (CTA) than in the naive or backward snails (control). *, P < 0.05; **, P < 0.01. Data are expressed as the mean ± SE. Modified from Kojima et al. (1997). Figure appears in colour on the Journal’s Web site.

RH155, and simulated the presentation of sucrose (i.e., the CS) with electrical stimulation of the median lip nerve. The median lip nerve transmits chemosensory signals of appetitive taste to the CNS. We optically detected a large number of spikes in several areas of the buccal ganglion after electrical stimulation of the median lip nerve. The effects of behavioral taste aversion training on the spike responses were examined in two areas of the buccal ganglion where the most active neural responses were seen. In one area that accounted for the N1M cells, the number of spikes after median lip nerve stimulation (i.e., the simulated CS) was significantly reduced in taste aversion trained snails compared wih control snails. In another area positioned between the buccal motor neurons (i.e., the B3 motor neuron and the B4 cluster cells), the evoked spike responses elicited by median nerve stimulation were unaffected in the taste aversion trained preparations. These data showed that the appetitive signal transmitted via the median lip nerve to the N1M cells is suppressed following CTA. This results in a decrease of the fictive feeding response. However, even with our optical recording technique, we still cannot rule out the possibility that changes in neuronal activity in other areas of the CNS occur with taste aversion training.

Memory trace in the feeding central pattern generator in conditioned taste aversion As described above, the polysynaptic IPSP recorded in the N1M cells by activation of the CGCs in taste aversion trained snails was larger and lasted longer than the IPSP in control snails (Fig. 4). However, the neural circuit between the CGC and the N1M cell consists of two types of synaptic connections: (1) the excitatory monosynaptic connection from the CGC to the neuron 3 tonic Published by NRC Research Press

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Ito et al.

Fig. 5. Enhancement of spontaneous excitatory postsynaptic potential (EPSP) in the B3 motor neurons after taste aversion training in the great pond snail (Lymnaea stagnalis). (A) Schematic presentation of the neural circuitry underlying taste aversion training. The signals of sucrose (conditioned stimulus: CS) and KCl (unconditioned stimulus: US) are associated in the cerebral giant cells (CGCs). Rectangles and circles indicate interneurons and motor neurons, respectively. At synapses, open circles and solid circles indicate excitatory monosynaptic inputs and inhibitory monosynaptic inputs, respectively. The neuron 1 medial (N1M), neuron 2 (N2), and neuron 3 tonic (N3t) cells form part of the feeding central pattern generator (CPG). (B) Spontaneous EPSP in the B3 motor neurons. The EPSP recorded in the B3 motor neurons can be used for monitoring the changes in the N3t–N1M synaptic connection. The B3 EPSPs recorded from the taste aversion trained snails were significantly larger (one-way ANOVA, P < 0.05) than those observed for the backward conditioned and naive control snails. 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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atory synaptic connections between the CS pathway and the feeding command neurons (Fig. 5A). Next, we hypothesized that “taste aversion learning” would occur via a mechanism which was the inverse of the mechanism proposed for “appetitive conditioning”. That is, there would be an increase in N3t spiking after conditioning, and this increase in N3t firing would inversely correlate with a reduction in the conditioned fictive feeding response. We thus hypothesized that taste aversion learning in L. stagnalis is also due to the combined effects of reduced tonic inhibition and enhanced excitatory synaptic connections between the CS pathway and the feeding command neurons. However, because the N3t cells are too small to access consistently by standard sharp electrode recording techniques, in the present study the synaptic inputs from the CGCs to the N3t cells and those from the N3t cells to the N1M cells were inferred by monitoring the monosynaptic excitatory postsynaptic potential (EPSP) recorded in the large B1 and B3 motor neurons, respectively. The evoked monosynaptic EPSPs of the B1 motor neurons in the brains isolated from the taste aversion trained snails were identical to those in the control snails, whereas the spontaneous monosynaptic EPSPs recorded in the B3 motor neurons were significantly enlarged (Fig. 5B; Ito et al. 2012). These data suggested that, after taste aversion training, the monosynaptic inputs from the N3t cells to the follower neurons, including the N1M cells, are facilitated. That is, one of the neural correlates of CTA–LTM is an increase in neurotransmitter release from the N3t cells. We thus conclude that the N3t cells suppress the N1M cells in the feeding CPG, in response to the CS in L. stagnalis CTA.

Development of the life cycle and development of the learning ability

(N3t) cell and (2) the inhibitory monosynaptic connection from the N3t cell to the N1M cell (Fig. 5A). As a next step, we had to determine which synaptic connection is more changed following the acquisition of CTA. The recent studies on appetitive conditioning of feeding behavior in L. stagnalis by the Benjamin group made three points (Marra et al. 2010). (1) Tonic inhibition in the feeding network is provided by the N3t cell. This interneuron makes a monosynaptic inhibitory connection with the N1M cell. (2) There is a reduction in N3t spiking after appetitive conditioning, and this reduction in N3t firing inversely correlates with an increase in the conditioned fictive feeding response. (3) Computer simulation of N3t–N1M interactions suggests that changes in N3t firing are sufficient to explain the increase in the fictive feeding activity produced by appetitive conditioning. These data showed that appetitive conditioning of feeding behavior in L. stagnalis occurs because of the combined effects of reduced tonic inhibition and enhanced excit-

By using the taste aversion training procedures in L. stagnalis, we can assess the common changes that occur both development of the life cycle and development of the learning ability of this organism (Ono et al. 2002; Karasawa et al. 2008; Sunada et al. 2010a). We examined developmental changes in the acquisition and retention of CTA in L. stagnalis (Yamanaka et al. 1999). Our data showed that snails developed their ability to form CTA–LTM through the three critical stages: (1) stage 25 embryos (veliconcha) start to respond to appetitive sucrose, (2) stage 29 embryos just before hatching acquire CTA, but not LTM, and (3) immature snails with a 10 mm shell are able to learn and remember which foods can be safely eaten. That is, the development of learning ability in snails is coincident with the major changes in their life cycle. We then examined the relationship between the learning ability for CTA and the development of the CGC for CTA. Using Lucifer-yellow staining of the CGCs and Azan staining for the ganglion sections, we found that the CGCs mature at the early developmental stages and that the number of buccal and cerebral neurons in immature snails is similar to that seen in adult snails (Sadamoto et al. 2000). The immunoreactivity of serotonin, which is one of the main neurotransmitters employed in the feeding circuitry (Kemenes et al. 1989, 1997; Kemenes 1997; Hatakeyama and Ito 1999; Nakamura et al. 1999c; Kawai et al. 2011), was first observed in the CGCs at stage 29 (Yamanaka et al. 2000). After hatching, the neuropile of CGCs developed faster than other cells in the buccal and cerebral ganglia, resulting in their early innervation at the immature stage. Thus, the developmental changes in the CGCs correlate well with the ability to form CTA.

Identification of interneurons involved in the withdrawal response that affect the cerebral giant cells Although we have some evidence that there are input pathways onto the CGCs from higher order interneurons which mediate the Published by NRC Research Press

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withdrawal response elicited by the KCl stimulus (Nakamura et al. 1999a, 1999b), these interneurons have not been positively identified (Ferguson and Benjamin 1991a, 1991b). However, two identified neurons are good candidates. One is the pleural–buccal neuron (PlB) and the other is the right pedal dorsal 11 neuron (RPeD11). The PlB is FMRFamidergic and was reported to inhibit all the neurons in the feeding circuit, including protraction and retraction motor neurons, feeding CPG interneurons, buccal modulatory interneurons, and the CGCs (Alania et al. 2004, 2008). On the other hand, the Sakakibara group with the help of K. Lukowiak demonstrated that the RPeD11 sends an inhibitory input onto the CGCs (Sunada et al. 2012). The RPeD11, a well-known interneuron receiving sensory input from the right parietal dorsal 3 (RPD3) and sending output to the motor cluster neurons right pedal G (RPeG) and the right cerebral A (RCeA), exerts withdrawal behavior in response to multimodal noxious stimuli such as mechanical prodding, KCl application, and shadow presentation (Sunada et al. 2010b). An aversive stimulus to L. stagnalis employed in CTA as a US basically resulted in withdrawal behavior. The Sakakibara group’s studies on neuronal mechanisms in the CTA used sucrose application as the CS and weak mechanical prodding to the animal’s head as the US (Kawai et al. 2004b). After acquisition of learning, the conditioned animals responded to decreases in the feeding response against the CS application. The presentation of the US, irrespective of whether KCl application or mechanical prodding was used, increased excitability in the RPeD11. Even with the application of weak mechanical prodding, the RPeD11 was excited, thereby decreasing the feeding response. The strong excitation induced by positive current injection into the RPeD11 resulted in inhibition of the CGCs, as evidenced by such effects as a decrease in spontaneous firing activity. This inhibitory effect was transmitted to the CGCs via mono-chemical synapses. The isolated preparations with mouth, buccal, and esophageal ganglia may provide a common platform for the CTA in vitro conditioning model using sucrose application as the CS and current injection into the RPeD11 as the US (Sunada et al. 2012).

Future questions In our studies designed to determine how long CTA–LTM persists, it became apparent that snails continued to eat their normal diet of lettuce or similar leafy plants in their home aquaria while still exhibiting CTA–LTM. Thus, it was unclear what the relationship was between a CTA for a specific CS and other appetitive food stimuli. If snails can successfully differentiate between appetitive food stimuli, where in the CNS does this occur? Our previous experiments showed that snails can be differentially conditioned to avoid one appetitive CS following taste aversion training while continuing to be responsive to a different appetitive food CS that has not been paired in a forward manner with an aversive US (Sugai et al. 2006). That is, L. stagnalis can distinguish between tastes during CTA. The neurons responsible for taste discrimination may be located in the CNS and most probably exist upstream of the CGCs, but we have to carefully address this question and attempt to find neurons involved in taste discrimination in the L. stagnalis CNS in the future.

Conclusion Researchers investigating CTA in rats and other mammals are often surprised to see that relatively simple invertebrate model systems, such as L. stagnalis, are also capable of acquiring CTA. In fact, one author (Bernstein 1999) concluded from our data that the neural circuitry required for this learning is fairly primitive. However, we consider that this perceived weakness of the L. stagnalis model is actually an advantage (e.g., Murakami et al. 2013), because the use of simple invertebrate systems can provide answers to important basic questions before we move on to more complex mammalian systems.

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The molecular mechanisms that regulate serotonin release from the CGCs have also been clarified in L. stagnalis. The key players in the molecular cascades are cAMP, protein kinase A, cAMP response element binding protein, CCAAT/enhancer binding protein, and serotonin 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 the amount of serotonin released from the CGCs plays an important role in CTA. These cascades will be reviewed elsewhere.

Acknowledgements This work was supported by KAKENHI from the Japan Society for the Promotion of Science (JSPS No. 21657022) to E.I. and grants from the Canadian Institutes of Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada (NSERC) to K.L.

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