(1) Classical food‐reward conditioning

Classical appetitive food‐reward conditioning involves a temporal‐contingent repeated presentation of a neutral CS with a US that elicits feeding. In Lymnaea, the US results in a sequence of rhythmic and stereotyped feeding movements consisting of opening the mouth, rasping with the radula, and closing the mouth (Kemenes & Benjamin, ¹⁹⁸⁹). A sucrose solution is typically used as the US, but tactile, chemical, or visual cues can all be used as a CS (Kemenes & Benjamin, ^{1989, 1994}; Staras, Kemenes & Benjamin, ¹⁹⁹⁸). In one of the first associative learning LTM experiments using Lymnaea (see online Supporting Information, Appendix S1 for full details of experimental procedures used in conditioning experiments with Lymnaea), investigators employed a single‐trial learning procedure consisting of a single pairing of amyl acetate, which typically does not elicit feeding, as the CS, with sucrose used as the US (Fig. S1). After the single‐trial training session (i.e. CS–US pairing), application of the CS alone in the memory test induced feeding behaviour, whereas feeding behaviour in response to the CS was not observed in any of the control groups, demonstrating associative learning (Alexander et al., ¹⁹⁸⁴). This single CS–US pairing was sufficient to create a LTM trace that persisted for at least 15days (Alexander et al., ¹⁹⁸⁴).

Single‐trial conditioning of feeding using a visual cue (see Appendix S1 for methodological details) is also possible by adding the US (sucrose) to a black panel (the CS) (Andrew & Savage, ²⁰⁰⁰), which snails perceive using a lens capable of forming an image on the retina underwater (Seyer, ¹⁹⁹²). After conditioning, approach to the black panel elicited more rasping movements in trained snails. It was also demonstrated that snails can learn to discriminate the black panel (CS) from a grey pattern of equal luminance, exhibiting the behavioural response only with the CS (Andrew & Savage, ²⁰⁰⁰).

Using appetitive classical conditioning, researchers were able to elucidate sites in the snails’ central nervous system (CNS) involved in learning this task and the mechanisms used to store memory‐related representations. In particular, some hours after training, conditioning leads to a reduction in spike activity in N3t that, in turn, makes N1M more likely to respond to the SNs (Marra et al., ²⁰¹⁰). Thus, there is a switch of the network from an inactive to a CS‐evoked rhythmically active state (Staras et al., ²⁰⁰³). At the same time, the conditioning procedure induces delayed but persistent depolarization of the CGCs, that facilitates inputs of the SN‐to‐CBI excitatory synapse (Kemenes et al., ²⁰⁰⁶). Because delayed depolarization of the CGCs is known to be correlated with the establishment and the duration of LTM, it has been assumed that CGCs are involved in the maintenance of the late phase of LTM (Nikitin et al., ²⁰⁰⁸).

Memory consolidation after single‐trial chemical appetitive classical conditioning represents a dynamic process that offers the opportunity to study both at the behavioural and neurophysiological level so‐called ‘memory lapses’. During memory consolidation, multiple learning events often occur in rapid succession and competition between the various consolidating memories can emerge, resulting in memory lapses (Marra et al., ²⁰¹³). Thus, single‐trial food‐reward memory could result in the erasure of newly acquired information (retroactive interference) or previous learning can affect the success of the acquisition of a second memory (proactive interference) (Crossley et al., ²⁰¹⁹). Whether proactive or retroactive interference is activated depends on the timing of the second training and the underlying neuronal mechanisms. In particular, it was demonstrated that when new learning takes place during a stable period of the original memory, proactive interference only occurs if the two consolidating memories engage the same circuit mechanisms. On the other hand, when different circuits are used, both memories survive. New learning occurring during a labile period of consolidation instead promotes retroactive interference and the acquisition of the new memory.

The success of classical conditioning training depends on both internal (e.g. food deprivation/satiety) and external variables (e.g. water conditions) (Kemenes & Benjamin, ¹⁹⁹⁴; Murakami et al., ²⁰¹³). Regarding external variables, Kemenes & Benjamin (¹⁹⁹⁴) showed that snails trained in a novel environment had better appetitive learning performance compared to animals trained in the water of their home tanks (i.e. a familiar environment). Moreover, the learning‐stimulating effect of the novel environment was enhanced if snails were food‐deprived before testing, whereas it was strongly suppressed in snails fed ad libitum, suggesting a reciprocal interaction between internal and external variables (Kemenes & Benjamin, ¹⁹⁹⁴).

In pivotal studies of reward conditioning, Audesirk et al. (¹⁹⁸²) demonstrated that although both ‘young’ and ‘old’ starved snails acquired the association between food and the chemo‐stimulus, ‘old’ snails had worse memory performance compared to ‘young’ ones and required multi‐trial experiments. ‘Old’ snails fed ad libitum before training failed to learn, while their younger counterparts acquired the association, but expressed the learned response only after a period of food deprivation (Audesirk et al., ¹⁹⁸²). Further studies demonstrated that the acquisition of appetitive memory was not affected by ageing, but that memory retention and consolidation become progressively impaired with advancing age (Hermann et al., ²⁰⁰⁷). Using appetitive classical conditioning, age‐associated learning and memory deficits were associated with declining electrophysiological excitability of the CGCs, most likely reflecting a selective deficit in the activity‐dependent regulation of gene transcription, possibly through CREB‐dependent mechanisms, as demonstrated in other models (Hermann et al., ²⁰⁰⁷; Pirger et al., ²⁰¹⁰; Scutt et al., ²⁰¹⁵). Memory decline in aged Lymnaea is consistent with the age‐dependent impairment in learning and memory functions observed in many animal species, including humans, where the molecular, cellular, and neural network functions show a decline of neuronal experience‐dependent plasticity (Hermann et al., ²⁰⁰⁷). Together, these results indicate that the Lymnaea feeding system is a powerful tool for investigators attempting to understand the cellular and molecular correlates of age‐related declines in cognitive ability.

(1) Operant conditioning of aerial respiration behaviour

Lymnaea is a bimodal breather: under normoxic (i.e. eumoxia) conditions, gaseous exchange occurs via transpiration across the skin (cutaneous exchange). In hypoxic conditions [as often arise in their pond‐water environment], snails come to the water surface for aerial respiration, consisting of rhythmic opening and closing of the pneumostome (Boycott, ¹⁹³⁶; Jones, ¹⁹⁶¹). Aerial respiratory behaviour can thus be used in operant conditioning without affecting the survival of the animal, as they can obtain oxygen via cutaneous exchange (Lukowiak et al., ¹⁹⁹⁶). In a ground‐breaking series of experiments the neuronal basis of the CPG that underlies aerial respiration in Lymnaea was elucidated (Syed et al., ^{1990, 1992}), showing that a three‐neuron network was both sufficient and necessary for aerial respiratory behaviour. Few, if any, other neuronal circuits underlying an easily observable and tractable behaviour have been described, and this system has been used to investigate the neuronal mechanisms of learning and LTM.

Lukowiak et al. (¹⁹⁹⁶) demonstrated that the aerial respiration behaviour can be operantly conditioned by applying a relatively weak tactile stimulus to the pneumostome each time snails attempt to open it. This negative reinforcement causes the snail to close its pneumostome but does not cause the animal to withdraw its foot and mantle area (i.e. the whole‐animal withdrawal response). This learning undergoes consolidation into either ITM or LTM (Lukowiak et al., ²⁰⁰⁰).

ITM, and LTM can be differentially produced by altering the duration, number and intervals used in training protocols for this behaviour (Lukowiak et al., ²⁰⁰⁰). For example, an operant training procedure of two 45‐min training sessions with a 1‐h interval between training sessions, followed by a third 45‐min training session 18 h later was sufficient to produce an LTM persisting for at least 5days (McComb et al., ²⁰⁰²); five training sessions of 30 min with an inter‐session interval of at least 1 h over the course of 2.5days was required for the establishment of a LTM that persisted for at least 1 week; eight training sessions repeated over a four‐week period resulted in a LTM that persisted for at least 1 month (Lukowiak et al., ¹⁹⁹⁸). A LTM lasting for 48 h can be evoked with a single day of training, by exposing snails to three series of 15‐min training sessions with an inter‐training interval of at least 1 h (Fig. S3A), whilst a 30‐min interval between sessions resulted in associative learning but memory persistence for only ~3 h, designated as ITM by Lukowiak et al. (²⁰⁰⁰) (Fig. S3B) (Lukowiak et al., ²⁰⁰⁰; Smyth, Sangha & Lukowiak ²⁰⁰²). Moreover, Smyth, Sangha & Lukowiak (²⁰⁰²) demonstrated that if snails received the ITM training procedure followed by 5 h in their home aquaria, subsequent training with the LTM procedure led to a LTM that persisted for at least 72 h; 24 h longer than usually produced by this LTM training procedure on its own.

It was proposed that the ITM training procedure initiates the translation of pre‐existing mRNA into proteins that bring about the neuronal changes necessary for memory. Although 5 h after the ITM training session memory can no longer be demonstrated behaviourally, it appears that at a neuronal level, some changes persisted for longer constituting a ‘trace’ of memory that was sufficient to enhance LTM formation and maintenance. This mechanism was described as “gone but not forgotten” by Smyth et al. (²⁰⁰²). If LTM training occurred 8 h after ITM training, LTM augmentation did not occur, suggesting that this inter‐training interval was too long for the memory trace to be maintained (Smyth et al., ²⁰⁰²). These results indicate that if the ITM trace is still present during LTM conditioning, then the memory trace can be reconstructed from a pre‐formed framework, enabling it to last for longer, whereas if LTM training occurred after the ITM trace had disappeared then the neurons must build a new memory framework (Smyth et al., ²⁰⁰²).

It was also demonstrated that extinction training after ITM training did not increase memory persistence following subsequent LTM training (Smyth et al., ²⁰⁰²). Extinction of the ITM was achieved by placing ITM‐trained snails in the same hypoxic environment for 90 min without a reinforcing stimulus. The snails were then immediately subjected to LTM training, but the memory phenotype did not persist for longer than 48 h (Smyth et al., ²⁰⁰²). Because extinction is viewed as a form of learning that co‐exists with the previously learned behaviour, it was assumed that ‘ITM protein’ was used up or replaced with ‘extinction protein’, resulting in no LTM enhancement (Smyth et al., ²⁰⁰²).

Operant conditioning of aerial respiration clearly represents a versatile behavioural procedure that can be used to elucidate what Smyth et al. (²⁰⁰²) defined as the “lingering effects” of ITM on LTM persistence in the title of their paper. Future studies using this system could investigate the neuronal and molecular changes involved in LTM enhancement and whether these are conserved in invertebrate and vertebrate models.

Both ITM and LTM can be extinguished (McComb et al., ²⁰⁰²). In this study, snails received three training sessions followed by extinction training in the same temporal sequence as the operant conditioning training but without a reinforcing stimulus. LTM was not observed when tested the following day (McComb et al., ²⁰⁰²).

From a neuronal perspective, the CPG driving aerial respiration behaviour consists of three interneurons: right pedal dorsal 1 (RPeD1), visceral dorsal 4 (VD4) and (wide‐acting) input 3 (IP3). Spiking activity in RPeD1 initiates rhythmic patterns of spiking activity of IP3 that results in pneumostome opening through monosynaptic excitatory synaptic connections to visceral J (VJ) pneumostome opener motor neurons. On the contrary, monosynaptic excitatory connections exist from VD4 to VK pneumostome closer motor neurons (Syed et al., ¹⁹⁹⁰; Spencer et al., ¹⁹⁹⁹). After conditioning, the excitatory input from RPeD1 to IP3 is weakened, resulting in reduction of the rhythmic activity of the pneumostome (Spencer et al., ¹⁹⁹⁹).

Direct evidence for the role of RPeD1 in operant conditioning comes from soma‐ablated snails: when the soma of RPeD1 was ablated before training, the CPG is only capable of mediating ITM, with LTM is no longer observed (Scheibenstock et al., ²⁰⁰²; Sangha, McComb & Lukowiak, ^2003a ). These data indicate that ITM and LTM are not only distinct in a chronological and transcriptional manner, but are also different at the neuronal level (Braun & Lukowiak, ²⁰¹¹). Because ITM is dependent on new protein synthesis but not altered gene activity (Scheibenstock et al., ²⁰⁰²), it seems that the remaining functional neurites translate the pre‐existing messenger RNA (mRNA) into new proteins (Braun & Lukowiak, ²⁰¹¹). However, following ablation of the soma of RPeD1 after consolidation, snails could still access or retrieve a previously encoded memory, indicating that the soma was not essential for memory retention (Lukowiak et al., ²⁰⁰³; Sangha, Scheibenstock & Lukowiak, ^2003b ). Thus, studies in Lymnaea demonstrate for the first time that a single neuron can be a site (although not necessarily the only site) for LTM formation and storage, enabling direct investigations of interactions between a single cell and a neuronal network that are necessary for memory formation, reconsolidation and extinction (Sangha et al., ^{2003a , b , c} ).

Because extinction is due to new memory, ablation of the soma of RPeD1 after LTM formation could potentially prevent memory erasure (Sangha et al., ^{2003a , 2005}). Moreover, because ablating the soma of RPeD1 disrupts the reconsolidation of a LTM, the critical molecular processes occurring during both consolidation and reconsolidation not only require both new RNA and protein synthesis, but also that these take place in the same cell in Lymnaea. These data are consistent with studies in mammals, where both memory formation and reconsolidation use similar pathways and occur in the same brain regions as the original learning (Nader, Schafe & Le Doux, ²⁰⁰⁰; Abel & Lattal, ²⁰⁰¹). Sangha et al. (²⁰⁰⁵) showed that ‘forgetting’ was an active process requiring altered gene activity and new protein synthesis. Ablation of the soma of RPeD1 after LTM consolidation resulted in snails that did not forget (LTM persisted for significantly longer).

Operant conditioning of Lymnaea aerial respiratory behaviour represents a useful tool to explore how internal and external variables affect learning and to elucidate how the context in which the snails are trained and tested interferes with associative learning (Fig. S4; Haney & Lukowiak, ²⁰⁰¹). Haney & Lukowiak (²⁰⁰¹) thereby demonstrated the context specificity of learning and LTM in Lymnaea. By training snails in a hypoxic environment with a food odorant (carrot odour), LTM was demonstrated only if carrot odour was present also at the time of the memory test (Haney & Lukowiak, ²⁰⁰¹). Interestingly, in Lymnaea not only learning, but also extinction is context dependent. In particular, if extinction training was given in a different context from that in which the learning and memory occurred (e.g. hypoxic pond water versus food‐odorant context), then extinction did not occur (McComb et al., ²⁰⁰²). These data confirm that extinction is itself a form of learning that occludes the original learned behaviour.

Interestingly, Lukowiak et al. (²⁰⁰³) demonstrated that snails trained in one context (context 1) but experiencing a new context (context 2) during the reconsolidation period (i.e. during the time when memory is labile) showed memory for context 2. This was defined by the authors as “memory infidelity”, where “infidelity” means that snails develop a memory for something they did not undergo training for (Fig. S5). When snails were exposed to context 2 only during the reconsolidation period they incorporated that new context into LTM, even though it was not part of their training (i.e. memory infidelity). Although control snails experienced context 2 for the same interval as the experimental group, because the exposure occurred after the reconsolidation period, memory infidelity did not occur. This approach identified ‘unfaithful’ memory for the first time in a molluscan model system. Moreover, it was the first time that this process had been observed in a non‐declarative memory in any model system (Lukowiak et al., ²⁰⁰⁷). Considering that it is relatively easy to modify a memory after its reactivation, future studies could attempt to use this system to gain new insights into how memories are made, maintained and modulated.

The operant conditioning paradigm of aerial respiration has also been used to explore how memory formation in Lymnaea is affected by environmental stress. Environmental stressors can have a strong, and sometimes unpredictable, modulatory effect on learning and memory formation depending on the nature of the stressor and when it is encountered relative to a period of learning. In Lymnaea, as in vertebrates, stressors may block or enhance learning and memory formation (Lukowiak et al., ²⁰¹⁴).

We previously explored how different periods of food deprivation could alter the CTA LTM (Ito et al., ²⁰¹⁵). Studies using aerial operant conditioning also demonstrate a better ability to form memory in snails trained in the presence of kairomones of their crayfish predator (Cambarus spp.) (i.e. the crayfish effluent) (Forest et al., ²⁰¹⁶; see Fig. S6). Note, however, that the snails used by Forest et al. (²⁰¹⁶) were first trained as juveniles, which typically are not capable of forming LTM (McComb, Varshney & Lukowiak, ²⁰⁰⁵). Interestingly, when those snails were allowed to mature into adults during 4weeks in their home aquaria and were then trained using the ITM procedure in pond water they formed a LTM. Thus, training the juvenile snails in the presence of crayfish effluent enabled formation of LTM in response to the ITM procedure (Forest et al., ²⁰¹⁶).

Adult snails trained with the LTM procedure in the presence of crayfish effluent exhibited increased persistence of LTM, that lasted for at least 8 h, suggesting that predator‐augmented memory formation allows the memory to be recalled under a broader range of challenges (Forest et al., ²⁰¹⁶). Interestingly, this memory enhancement is associated with long‐lasting changes in RPeD1 (Orr & Lukowiak, ²⁰⁰⁸), suggesting that interspecific chemical communication can augment memory formation, inducing long‐lasting changes in this single neuron necessary for LTM formation. This research opens the possibility to investigate how ecologically relevant stressful stimuli can alter behaviour, learning and memory at the level of a single neuron (Dalesman & Lukowiak, ²⁰¹²). Enhanced LTM formation after training in the presence of crayfish effluent is not observed if snails are exposed to the serotonin blockers mianserin and methysergide, meaning that the perception of risk activates a neuronal mechanism that enhances memory formation through serotonergic modulation (Forest et al., ²⁰¹⁶). These data are consistent with previous studies in humans correlating increased serotoninergic tone with enhanced responses to anxiety‐related stimuli and memory consolidation (Inoue, Koyama & Yamashita, ¹⁹⁹³; Ji & Suga, ²⁰⁰⁷). Stress‐mediated memory enhancement may not necessarily be beneficial and can be exacerbated in pathologies such as post‐traumatic stress disorder (PTSD) and panic attacks (Lancaster et al., ²⁰¹⁶). Thus, Lymnaea represents a valid model system for elucidating how behaviourally relevant stressors can alter LTM formation and/or its persistence and may guide researchers towards possible therapeutic strategies for these psychiatric disorders.

In a recent study, Swinton et al. (²⁰¹⁹) demonstrated that snails can exhibit configural learning, a higher‐order form of learning and memory involving the ability to assign importance to different stimuli. In their protocol, snails simultaneously experienced the odours of crayfish effluent and carrot. Afterwards, animals were transferred for 2 h into their home aquarium where neither odour was present. Snails were then trained with a single training session lasting 30 min (ITM procedure) in hypoxic pond water containing carrot odour. Memory was tested 24 h later in hypoxic pond water and it was found that LTM was formed (Fig. S7). Enhanced LTM formation confirmed that the simultaneous exposure to the two stimuli together led carrot odour, which typically elicits feeding behaviour (Sugai et al., ²⁰⁰⁶), to acquire the properties of the predator kairomone in enhancing memory‐forming ability (Orr et al., ²⁰⁰⁷). This result did not occur when carrot odour was paired with boiled crayfish effluent, as boiling inactivated its predator‐signalling properties (Orr et al., ²⁰⁰⁷), nor with simultaneous exposure to both odours for only 10 min. Finally, enhanced LTM formation was not observed if training occurred in pond water in the absence of carrot odour. These data show that snails not only possess the ability to consider the individual components of a stimulus but also can form a relationship between the two components that is treated differently from the sum of these stimuli. Interestingly, simultaneous exposure for 45 min to both odours was shown to alter the feeding responses that typically occur when snails are exposed to carrot odour (Swinton et al., ²⁰¹⁹). At the end of the conditioning procedure carrot odour no longer elicited a significant increase in the feeding response, suggesting that after pairing of the two odours snails can assign a new meaning to carrot odour that now signals ‘fear’ rather than ‘food’. In other words, carrot odour acquired a new motivational state (i.e. fear) as opposed to its intrinsic motivational state (i.e. enhanced rasping).

Experience with predators not only shapes learning to guide value‐based behaviours, but that value can also be transferred between stimuli that perceptually or conceptually resemble one another (Orr et al., ²⁰⁰⁷). Such stimulus generalization confers important adaptive advantages allowing the animal to decipher the meaning of important stimuli and to generalize the context of a predator encounter to other environmental conditions. Thus, it is not surprising that this learning process has been conserved across taxa (Swinton et al., ²⁰¹⁹).

The experiments and results described above highlight the power of behavioural training in Lymnaea to elucidate the neuronal–behavioural crosstalk involved in learning and to study the role of context, stress and internal variables on memory formation and retrieval. The ability of snails to undergo configural learning confirms their potential in neuroscience and behavioural research.

The authors acknowledge the great body of data on various aspects of L. stagnalis learning and memory produced by numerous colleagues over decades. We are sincerely grateful to them for their fruitful research. Open access funding enabled and organized by Projekt DEAL.