An automated learning apparatus for classical conditioning of Lymnaea stagnalis.

BACKGROUND: The pond snail Lymnaea stagnalis is capable of taste avoidance classical conditioning (TAC) with sucrose as the conditional stimulus (CS) and mechanical prodding as the unconditional stimulus (US). After successful training, feeding behavior is significantly suppressed in response to CS presentation. NEW METHOD: An automated apparatus is described for the training of multiple snails up to 10 snails at the same time. The new apparatus employs an electrical shock obtained from a 9-V dry cell to deliver a consistent and effective current amplitude of 0.4µA (i.e., the US). RESULTS: Using this apparatus, 10 snails can be conditioned simultaneously. We found that the optimal parameters to result in both short (STM) and long-term memory (LTM) were 15 paired presentations of the CS and US with a 5-min inter-trial interval (ITI) and 0.2-s current duration. However, both STM and LTM were observed with other ITIs tested. Successful TAC with only a single pairing of the CS-US occurred with a CS of 100mM sucrose solution for 60s followed by a US of 9V with 0.4µA for 5s. COMPARISON WITH EXISTING METHOD: The use of automated training apparatus for TAC will enable us to better examine the relationship between strength of CS and US.

The Yerkes-Dodson law and appropriate stimuli for conditioned taste aversion in Lymnaea.

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.

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 animal's 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.

Spaced taste avoidance conditioning in Lymnaea.

We succeeded in taste avoidance conditioning with sucrose as the conditional stimulus (CS) and an electrical stimulus (∼1000V, 80µA) as the unconditional stimulus (US). 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) persisting for at least one week. However, while STM was elicited with 5, 8, 10, and 20 paired presentations of the CS-US on a single day, LTM was not. We found, however, that if we inserted a 3h interval between a first and a second set of CS-US pairings that both 8 and 20 paired CS-US presentations on a single day was now sufficient to cause LTM formation. Exposing snails to bryostatin before or during training enhanced LTM formation such that 8 paired presentations of the CS-US resulted in LTM.

Morphological and physiological characteristics of dermal photoreceptors in Lymnaea stagnalis.

Dermal photoreceptors located in the mantle of Lymnaea stagnalis were histologically and physiologically characterized. Our previous study demonstrated that the shadow response from dermal photoreceptors induces the whole-body withdrawal response. Through the interneuron, RPeD11, we detected that the light-off response indirectly originated from a dermal photoreceptor. Previous observations, based on behavioral pharmacology, revealed that cyclic guanosine monophosphate acts as a second messenger in the dermal photoreceptor. Furthermore, gastropods possess dermal photoreceptors containing rhodopsin, as a photopigment, and another photo-sensitive protein, arrestin, responsible for terminating the light response. Thus, we chose three antibodies, anti-cGMP, anti-rhodopsin, and anti-ß-arrestin, to identify the dermal photoreceptor molecules in Lymnaea mantle. Extracellular recording, using a suction electrode on the mantle, revealed a light off-response from the right parietal nerve. Overlapping structures, positive against each of the antibodies, were also observed. Numerous round, granular particles of 3-47 µm in diameter with one nucleus were distributed around pneumostome and/or inside the mantle. The cells surrounding the pneumostome area, located 10 µm beneath the surface, tended to have smaller cell soma ranging from 3 to 25 µm in diameter, while cells located in other areas were distributed uniformly inside the mantle, with a larger diameter ranging from 12 to 47 µm. The histological examination using back-filing Lucifer Yellow staining of the right parietal nerve with the three dermal photoreceptor antibodies confirmed that these overlapping-stained structures were dermal photoreceptors in Lymnaea.

Electrophysiological characteristics of feeding-related neurons after taste avoidance Pavlovian conditioning in Lymnaea stagnalis.

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.

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.

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.

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.