Hiroshi Sunada

Conditioned taste aversion with sucrose and tactile stimuli in the pond snail Lymnaea stagnalis

A new form of taste aversion conditioning was established in the pond snail Lymnaea stagnalis. An associative memory, lasting 24h, was produced in the pond snail with 20 pairings of 100 mM sucrose as the conditioned stimulus (CS) and mechanical stimulation to the head as the unconditioned stimulus (UCS). Animals exposed to reverse pairings of the CS and UCS failed to learn the association. The learning was characterized by a shift in the response to the UCS from a whole-body withdrawal response to the cessation of feeding behavior.

Involvement of Insulin-Like Peptide in Long-Term Synaptic Plasticity and Long-Term Memory of the Pond Snail Lymnaea stagnalis

The pond snail Lymnaea stagnalis is capable of learning taste aversion and consolidating this learning into long-term memory (LTM) that is called conditioned taste aversion (CTA). Previous studies showed that some molluscan insulin-related peptides (MIPs) were upregulated in snails exhibiting CTA. We thus hypothesized that MIPs play an important role in neurons underlying the CTA–LTM consolidation process. To examine this hypothesis, we first observed the distribution of MIP II, a major peptide of MIPs, and MIP receptor and determined the amounts of their mRNAs in the CNS. MIP II was only observed in the light green cells in the cerebral ganglia, but the MIP receptor was distributed throughout the entire CNS, including the buccal ganglia. Next, when we applied exogenous mammalian insulin, secretions from MIP-containing cells or partially purified MIPs, to the isolated CNS, we observed a long-term change in synaptic efficacy (i.e., enhancement) of the synaptic connection between the cerebral giant cell (a key interneuron for CTA) and the B1 motor neuron (a buccal motor neuron). This synaptic enhancement was blocked by application of an insulin receptor antibody to the isolated CNS. Finally, injection of the insulin receptor antibody into the snail before CTA training, while not blocking the acquisition of taste aversion learning, blocked the memory consolidation process; thus, LTM was not observed. These data suggest that MIPs trigger changes in synaptic connectivity that may be correlated with the consolidation of taste aversion learning into CTA–LTM in the Lymnaea CNS.

Environmentally relevant stressors alter memory formation in the pond snail Lymnaea

Stress alters adaptive behaviours such as learning and memory. Stressors can either enhance or diminish learning, memory formation and/or memory recall. We focus attention here on how environmentally relevant stressors alter learning, memory and forgetting in the pond snail, Lymnaea stagnalis. Operant conditioning of aerial respiration causes associative learning that may lead to long-term memory (LTM) formation. However, individual ecologically relevant stressors, combinations of stressors, and bio-active substances can alter whether or not learning occurs or memory forms. While the behavioural memory phenotype may be similar as a result of exposure to different stressors, how each stressor alters memory formation may occur differently. In addition, when a combination of stressors are presented it is difficult to predict ahead of time what the outcome will be regarding memory formation. Thus, how combinations of stressors act is an emergent property of how the snail perceives the stressors.

Increase in excitability of RPeD11 results in memory enhancement of juvenile and adult Lymnaea stagnalis by predator-induced stress

Memory consolidation following learning is a dynamic process. Thus, long-term memory (LTM) formation can be modulated by many factors, including stress. We examined how predator-induced stress enhances LTM formation in the pond snail Lymnaea stagnalis at both the behavioral and electrophysiological levels. Training snails in crayfish effluent (CE; i.e., water from an aquarium containing crayfish) significantly enhanced LTM. That is, while memory persists for only 3h in adult control experiments following a single 0.5-h training session in pond water in which the pneumostome receives a contingent tactile stimulus to the pneumostome; when the snails are trained in CE, the memory persists for at least 24h. In juveniles, the data are more dramatic. Juveniles are unable to form LTM in pond water, but form LTM when trained in CE. Here we examined whether juvenile snails form LTM following a one-trial training procedure (1TT). Following the 1TT procedure (a single-trial aversive operant conditioning training procedure), juveniles do not form LTM, unless trained in CE. Concomitantly, we observe changes in the excitability of RPeD11, a key neuron mediating the whole snail withdrawal response, which may be a neural correlate of enhanced memory formation.

The shadow-induced withdrawal response, dermal photoreceptors, and their input to the higher-order interneuron RPeD11 in the pond snail Lymnaea stagnalis

SUMMARY The shadow-induced withdrawal response in Lymnaea stagnalis is mediated by dermal photoreceptors located on the foot, mantle cavity, and skin around the pneumostome area. Here, we determined whether we could obtain a neural correlate of the withdrawal response elicited by a shadow in a higher-order central neuron that mediates withdrawal behavior. We measured the electrophysiological properties of the higher-order interneuron Right Pedal Dorsal 11 (RPeD11), which has a major role in Lymnaea withdrawal behavior. In semi-intact preparations comprising the circumesophageal ganglia, the mantle cavity and the pneumostome, but not the foot and eyes, a light-on stimulus elicited a small short-lasting hyperpolarization and a light-off stimulus elicited a depolarization of RPeD11. We also determined that dermal photoreceptors make a monosynaptic contact with RPeD11. The dermal photoreceptor afferents course to the circumesophageal ganglia via the anal and genital nerves to the visceral ganglion, and/or via the right internal and external parietal nerves to the parietal ganglion. Finally, in addition to responding to photic stimuli, RPeD11 responds to both mechanical and chemical stimuli delivered to the pneumostome.

High voltage with little current as an unconditional stimulus for taste avoidance conditioning in Lymnaea stagnalis.

A new and better taste avoidance conditioning paradigm for Lymnaea has been developed that replaces the previously used tactile unconditional stimulus (US) with an brief electrical stimulus (1000V, 80μA), while continuing to use a sucrose application to the lips as the conditional stimulus (CS). With 15 paired CS-US presentations on a single day, we were able to elicit both short-term memory (STM) and long-term memory (LTM). The LTM persisted for at least one week. While STM was elicited with 5, 8, or 10 paired presentations of the CS-US on a single day, LTM was not. The new US used here was more consistent than the previously used US, and this stimulus consistency may explain why 15 paired CS-US presentations now result in LTM formation.

Combining Stressors That Individually Impede Long-Term Memory Blocks All Memory Processes

The effects of stress on memory are typically assessed individually; however, in reality different stressors are often experienced simultaneously. Here we determined the effect that two environmentally relevant stressors, crowding and low calcium availability, have on memory and neural activity following operant conditioning of aerial respiration in the pond snail, Lymnaea stagnalis. We measured aerial breathing behaviour and activity of a neuron necessary for memory formation, right pedal dorsal 1 (RPeD1), in the central pattern generator (CPG) that drives aerial respiration in untrained animals, and assessed how these traits changed following training. In naïve animals both crowding and combined stressors significantly depressed burst activity in RPeD1 which correlated with a depression in aerial breathing behaviour, whereas low calcium availability had no effect on RPeD1 activity. Following training, changes in burst activity in RPeD1 correlated with behavioural changes, decreasing relative to their naïve state at 3 h and 24 h in control conditions when both intermediate-term memory (ITM: 3 h) and long-term memory (LTM: 24 h) are formed, at 3 h but not 24 h when exposed to individual stressors when only ITM is formed, and did not change in combined stressors (i.e. when no memory is formed). Additionally, we also found that Lymnaea formed short-term memory (STM: 10 min) in the presence of individual stressors or under control conditions, but failed to do so in the presence of combined stressors. Our data demonstrate that by combining stressors that individually block LTM only we can block all memory processes. Therefore the effects of two stressors with similar individual affects on memory phenotype may be additive when experienced in combination.

Protein kinase C mediates memory consolidation of taste avoidance conditioning in Lymnaea stagnalis

In Lymnaea stagnalis, in order to obtain a 10 min short-term memory (STM) of taste avoidance conditioning (TAC) at least 10 paired presentations of a conditioned stimulus (CS), sucrose, and an unconditioned stimulus (US), tactile stimulation to the animals head, are required. Pre-exposure of snails to the protein kinase C (PKC) α and ε activator bryostatin (Bryo) facilitated STM formation in that only 5 paired CS-US trials were required. Typically 20 paired presentations of the CS-US are required for formation of STM and LTM. However, 20 paired presentations do not result in STM or LTM if snails are pre-incubated with a PKC inhibitor, Ro-32-0432. We also found that LTM lasting longer than 48 h was acquired with Bryo incubation for 45 min even after termination of the conditioning paradigm. These data suggest that activation of the α and ε isozymes of PKC is crucially involved in the formation of LTM and provide further support for a mechanism that has been conserved across the evolution of species ranging from invertebrate molluscs to higher mammals.

Critical period of memory enhancement during taste avoidance conditioning in Lymnaea stagnalis.

The present study investigated the optimal training procedure leading to long-lasting taste avoidance behavior in Lymnaea. A training procedure comprising 5 repeated pairings of a conditional stimulus (CS, sucrose), with an unconditional stimulus (US, a tactile stimulation to the animal’s head), over a 4-day period resulted in an enhanced memory formation than 10 CS-US repeated pairings over a 2-day period or 20 CS-US repeated pairings on a single day. Backward conditioning (US-CS) pairings did not result in conditioning. Thus, this taste avoidance conditioning was CS-US pairing specific. Food avoidance behavior was not observed following training, however, if snails were immediately subjected to a cold-block (4°C for 10 min). It was critical that the cold-block be applied within 10 min to block long-term memory (LTM) formation. Further, exposure to the cold-block 180 min after training also blocked both STM and LTM formation. The effects of the cold-block on subsequent learning and memory formation were also examined. We found no long lasting effects of the cold-block on subsequent memory formation. If protein kinase C was activated before the conditioning paradigm, snails could still acquire STM despite exposure to the cold-block.

Function of insulin in snail brain in associative learning

Insulin is well known as a hormone regulating glucose homeostasis across phyla. Although there are insulin-independent mechanisms for glucose uptake in the mammalian brain, which had contributed to a perception of the brain as an insulin-insensitive organ for decades, the finding of insulin and its receptors in the brain revolutionized the concept of insulin signaling in the brain. However, insulin’s role in brain functions, such as cognition, attention, and memory, remains unknown. Studies using invertebrates with their open blood-vascular system have the promise of promoting a better understanding of the role played by insulin in mediating/modulating cognitive functions. In this review, the relationship between insulin and its impact on long-term memory (LTM) is discussed particularly in snails. The pond snail Lymnaea stagnalis has the ability to undergo conditioned taste aversion (CTA), that is, it associatively learns and forms LTM not to respond with a feeding response to a food that normally elicits a robust feeding response. We show that molluscan insulin-related peptides are up-regulated in snails exhibiting CTA–LTM and play a key role in the causal neural basis of CTA–LTM. We also survey the relevant literature of the roles played by insulin in learning and memory in other phyla.