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...

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Endoreplication is DNA synthesis without cell division. Giant neurons observed in the brains of mollusks are thought to be generated as a result of DNA endoreplication. It has been hypothesized that neuronal size becomes larger in parallel with an increase in body size and that DNA endoreplication is involved in this process to meet the increasing demand for macromolecules in neurons. There is, however, no experimental evidence for this hypothesis to date. In the present study, we investigated the following quantitatively: (1) the size of the brain and each ganglion, (2) the size of identified neurons, (3) the total number of neurons undergoing DNA endoreplication, (4) the total number of the neurons containing a cardioexcitatory peptide, and (5) the gene expression level per neuron, using terrestrial slugs whose body growth was regulated through the amount of food supplied in the laboratory. The body growth was accompanied by increases in the sizes of both neurons and ganglia and triggered more frequent DNA endoreplication events in each ganglion of the growth-promoted slugs, without increasing the total number of neurons. Increase in the neuronal size also involved the increase in the amount of transcripts expressed in a single neuron. This is the first quantitative evidence showing that the DNA endoreplication, neuronal size, and gene expression are increased concomitantly with body growth in adult mollusks.

Food deprivation for 1 day in the pond snail Lymnaea stagnalis before aversive classical conditioning results in optimal conditioned taste aversion (CTA) and long-term memory (LTM) formation, whereas 5-day food deprivation before training does not. We hypothesize that snails do in fact learn and form LTM when trained after prolonged food deprivation, but that severe food deprivation blocks their ability to express memory. We trained 5-day food-deprived snails under various conditions, and found that memory was indeed formed but is overpowered by severe food deprivation. Moreover, CTA-LTM was context dependent and was observed only when the snails were in a context similar to that in which the training occurred.