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...

The pond snail Lymnaea stagnalis can learn conditioned taste aversion and then consolidate it into long-term memory (LTM). A high-voltage electric shock was used as the unconditioned stimulus, where we have previously used KCl. We varied the strength of both the conditioned and unconditioned stimuli to determine whether the so-called Yerkes-Dodson law prevailed. This is an empirical relationship between the state of arousal and LTM formation, showing that there is an optimal level of arousal leading to memory formation. However, too little or too much arousal results in poorer LTM. We found here that the most appropriate stimuli to use in taste aversion training in Lymnaea were a 10 mmol l −1 sucrose solution as the conditioned stimulus and a 3 s electric shock as the unconditioned stimulus.

No abstract

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.

Taste avoidance conditioning (TAC) was carried out on the pond snail, Lymnaea stagnalis. The conditional stimulus (CS) was sucrose which elicits feeding behavior; while the unconditional stimulus (US) was a tactile stimulus to the head which causes feeding to be suppressed. The neuronal circuit that drives feeding behavior in Lymnaea is well worked out. We therefore compared the physiological characteristics on 3 classes of neurons involved with feeding behavior especially in response to the CS in conditioned vs. control snails. The cerebral giant cell (CGC) modulates feeding behavior, N1 medial neuron (N1M) is one of the central pattern generator neurons that organizes feeding behavior, while B3 is a motor neuron active during the rasp phase of feeding. We found the resting membrane potential in CGC was hyperpolarized significantly in conditioned snails but impulse activity remained the same between conditioned vs. control snails. There was, however, a significant increase in spontaneous activity and a significant depolarization of N1M’s resting membrane potential in conditioned snails. These changes in N1M activity as a result of training are thought to be due to withdrawal interneuron RPeD11 altering the activity of the CGCs. Finally, in B3 there was: 1) a significant decrease in the amplitude and the frequency of the post-synaptic potentials; 2) a significant hyperpolarization of resting membrane potential in conditioned snails; and 3) a disappearance of bursting activity typically initiated by the CS. These neuronal modifications are consistent with the behavioral phenotype elicited by the CS following conditioning.