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. IntroductionFormation of long-term memory (LTM) after associative learning is dependent on both protein synthesis and altered gene activity in neurons that play a critical role in memory formation (Inda et al., 2005;Lee et al., 2008;Rosenegger et al., 2010). The pond snail Lymnaea stagnalis is a good model in which to elucidate the causal mechanisms that underlie LTM formation (Ito et al., 1999(Ito et al., , 2012a Sakakibara, 2006;Nikitin et al., 2008;Kemenes and Benjamin, 2009). In conditioned taste aversion (CTA), a form of associative learning, an appetitive stimulus (sucrose) is used as the conditioned stimulus (CS), and an aversive stimulus (KCl) is used as the unconditioned stimulus (US). The CS increases the feeding response in snails, whereas the US inhibits feeding. In CTA training, the CS is paired with the US. After repeated paired presentations, the CS no longer elicits the feeding response, and this aversive conditioning persists as LTM (Kojima et al., 1996).We identified candidate genes necessary for the establishment of CTA-LTM in Lymnaea and found that some genes were upregulated while others were downregulated . Some of the upregulated genes after LTM consolidation were the molluscan insulin-related peptide (MIP I, II, and others) genes. However, it is unclear whether MIPs are necessary for memory consolidation, and if they are, what is their role in the consolidation process.Peptide purification of MIP I-III and V and the additi...

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. KEY WORDS: Operant conditioning, Environmental impact, Memory formation, Long-term memory, Social snails IntroductionThe ability of animals to learn and remember enables them to adapt to environmental changes, and directly affects 'fitness'. Its common knowledge that stress affects learning and memory. This has been known in the literature since Bacon (Bacon, 1620) and is exemplified in the 'Yerkes-Dodson Law' or as we call it the 'Goldilocks Rule' (Yerkes and Dodson, 1908) (Fig. 1). Too much or too little stress impedes long-term memory (LTM) formation, while 'just the right amount' enhances LTM. Organisms are thought to 'decide' to only expend the 'neuronal cost' (e.g. altered gene activity and new protein synthesis) necessary to form LTM to 'relevant' events. Relevancy is in part determined by the level of stress perceived at the time of learning. Because memory is dynamic, stress can modify memory (e.g. false memory and post-traumatic stress syndrome) (Shors, 2004; Kim and Diamond, 2002;Lukowiak et al., 2003;Lukowiak et al., 2008;Lukowiak et al., 2010). Here we will focus on environmentally relevant stressors that alter memory formation in Lymnaea.Hans Selye, the 'father of stress research' stated: 'Everyone knows what stress is and nobody knows what it is ' (Selye, 1973). Stress is defined here as a state that requires a physiological, psychological or behavioural readjustment or modification in order to maintain the well being of the organism. However, the same stimulus may be perceived as a stressor for one organism but not for another, or may be perceived as a stressor only at certain times and not at others in the same organism. The effects of stress on learning and memory sometimes yield contradictory results (Shors, 2004). Because of the complexities of the brain and the multitude of behaviours tested, in addition to the different ways in which stressors act, disagreement in the literature on the role that stress plays in learning and memory formation is not too surprising. Using a simpler model system and...

Stress alters the ability to form, recall and maintain memory according to the Yerkes-Dodson/Hebb (YDH) law. The effects of environmentally relevant stressors, such as low environmental calcium and crowding, on learning and memory have previously been described in a laboratory-reared 'average' strain of Lymnaea stagnalis (i.e. the Dutch strain) as well as two strains of freshly collected L. stagnalis with enhanced memory formation abilities (i.e. 'smart' snails). Here, we use L. stagnalis to study the effects of other environmentally relevant stressors on memory formation in two other strains of freshly collected snails, one 'smart' and one 'average'. The stressors we examined are thermal, resource restriction combined with food odour, predator detection and, for the first time, tissue injury (shell damage). We show that the same stressor has significantly different effects on memory formation depending on whether snails are 'smart' or 'average'. Specifically, our data suggest that a stressor or a combination of stressors act to enhance memory in 'average' snails but obstruct memory formation in 'smart' snails. These results are consistent with the YDH law and our hypothesis that 'smart' snails are more easily stressed than 'average' snails.

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Environmentally relevant stressors alter the memory-forming process in Lymnaea following operant conditioning of aerial respiration. One such stressor is heat. Previously, we found that following a 1 h heat shock, long-term memory (LTM) formation was enhanced. We also had shown that the heat stressor activates at least two heat shock proteins (HSPs): HSP40 and HSP70. Here, we tested two hypotheses: (1) the production of HSPs is necessary for enhanced LTM formation; and (2) blocking DNA methylation prevents the heat stressor-induced enhancement of LTM formation. We show here that the enhancing effect of the heat stressor on LTM formation occurs even if snails experienced the stressor 3 days previously. We further show that a flavonoid, quercetin, which inhibits HSP activation, blocks the enhancing effect of the heat stressor on LTM formation. Finally, we show that injection of a DNA methylation blocker, 5-AZA, before snails experience the heat stressor prevents enhancement of memory formation.