Reconsolidation of a long-term memory in Lymnaea requires new protein and RNA synthesis and the soma of right pedal dorsal 1.

Reconsolidation of a long-term memory (LTM) in the snail Lymnaea stagnalis can be disrupted by cooling, an RNA synthesis blocker (actinomycin D), and by specifically ablating the soma of a cell we know is a site of LTM consolidation (right pedal dorsal 1, RPeD1). Aerial respiratory behavior was conditioned operantly by applying a gentle tactile stimulus to the pneumostome area (the respiratory orifice) every time the snail began to open its pneumostome to perform aerial respiration. This resulted in a reduction of this behavior while leaving cutaneous respiration intact. One week after training one-half of the animals received a memory reactivation session, which was similar to the original training (i.e., animals received reinforcement). All animals then received 1 hr of cooling, an injection of actinomycin D or saline, or the soma ablation procedure. This was followed by a test for savings 4 hr or 4 d later, which was also similar to the original training. Only those animals that received both the memory reactivation session and the treatment showed memory impairment during the test for savings. That is, the impairment was contingent on memory reactivation. These data indicate that reconsolidation requires both new RNA and protein synthesis to stabilize a reactivated memory, and it demonstrates that the soma of RPeD1, a cell that we have shown previously to be required in the consolidation of an LTM, is necessary for reconsolidation. These data suggest that the critical molecular processes occurring during both consolidation and reconsolidation transpire in the same cell in Lymnaea.

Extinction requires new RNA and protein synthesis and the soma of the cell right pedal dorsal 1 in Lymnaea stagnalis.

Lymnaea stagnalis were operantly conditioned to not perform aerial respiratory behavior. This learned response was subsequently extinguished. Here, we show that spaced extinction training is more effective than massed extinction training, in addition to the occurrence of spontaneous recovery. We also find evidence of a critical period within the first hour after extinction training in which new RNA and protein synthesis must occur for a memory of extinction training to be established. The memory for extinction training can also be extended using cooling and by preventing aerial respiration from occurring after extinction training. In addition, we demonstrate that memory formation of extinction training requires the soma of the cell right pedal dorsal 1, a cell that we have previously shown to be necessary for long-term memory consolidation and reconsolidation. This finding implies that the events that lead to the formation of extinction memory occur in the same cell that is responsible for long-term memory of operant conditioning. All of these data are consistent with the hypothesis that, during extinction, a new associative memory is being formed and that this new memory covers up, but does not abolish, the "old" memory.

Impairing forgetting by preventing new learning and memory.

Two causes of forgetting have been promulgated: memory trace decay and retroactive interference. The authors show that forgetting is an active process requiring both new learning and memory. In the present (1)Lymnaea model system, prevention of new learning of a conflicting association, inhibition of memory consolidation, or Right Pedal Dorsal 1 soma ablation, which blocks LTM formation, are all potent means to prevent forgetting. Thus procedures that alter the ability to learn or form memory of a new conflicting aerial respiratory association prevent forgetting of a learned associative behavior. These results are the 1st demonstration in any model system that forgetting requires the soma of a single neuron.

A molluscan model system in the search for the engram.

A 3-neuron central pattern generator, whose sufficiency and necessity has been directly demonstrated, mediates aerial respiratory behaviour in the pond snail, Lymnaea stagnalis. This behaviour can be operantly conditioned, and this associative learning is consolidated into long-lasting memory. Depending on the operant conditioning training procedure used the learning can be consolidated into intermediate term (ITM) or long-term memory (LTM). ITM persists for only 2-3 h, whilst LTM persists for days to weeks. LTM is dependent on both altered gene activity and new protein synthesis while ITM is only dependent on new protein synthesis. We have now directly established that one of the 3-CPG neurons, RPeD1, is a site of LTM formation and storage. We did this by ablating the soma of RPeD1 and leaving behind a functional primary neurite capable of mediating the necessary synaptic interactions to drive aerial respiratory behaviour by the 3-neuron CPG. However, following soma ablation the neuronal circuit is only capable of mediating learning and ITM. LTM can no longer be demonstrated. However, if RPeD1's soma is ablated after LTM consolidation memory is still present. Thus the soma is not needed for the retention of LTM. Using a similar strategy it may be possible to block forgetting.

Intermediate and long-term memories of associative learning are differentially affected by transcription versus translation blockers in Lymnaea.

Aerial respiratory behaviour in the pond snail, Lymnaea stagnalis, can be operantly conditioned. This associative learning then undergoes consolidation into a long-lasting memory which, depending on the training procedure used, causes intermediate-term memory (ITM; lasting 3 h) or long-term memory (LTM; lasting >6 h) to be formed. We determined the differential susceptibility of these two forms of memory to translation and transcription blockers. The injection of a translation blocker, Anisomycin, 2.5 h before training prevents the establishment of both ITM and LTM. On the other hand, injection of the transcription blocker Actinomycin D, 2.5 h before training, did not prevent the establishment of ITM, but did, however, prevent LTM formation. Thus in Lymnaea, following associative learning, both ITM and LTM are dependent on new protein synthesis. ITM appears to be dependent on protein synthesis from preexisting transcription factors, whilst LTM is dependent on protein synthesis from new transcription messages.

Associative learning and memory in Lymnaea stagnalis: how well do they remember?

The search for 'the how and the where' of memory formation in the brain, the engram, is still one of the unattained 'Holy Grails' of neuroscience. Over the years, various paths have been trodden in attempts to attain this goal, and while tantalizing glimpses appear now and then on the scientific horizon, the Grail still has not been grasped. One of the paths that investigators have walked is the invertebrate 'model system' approach. Some invertebrates possess relatively simple nervous systems that mediate relatively simple behaviours that are both interesting and trainable. In this commentary, we would like to shed light on a relatively new player, the pond snail Lymnaea stagnalis L., that is being used in the quest to illuminate 'the how and the where' the nervous systems encode and store memory. We will show that it is possible to demonstrate that a single neuron is a site of memory formation and storage for a form of associative learning in this lowly snail. It may be that the Grail is a little closer to being grasped.

The Soma of RPeD1 must be present for long-term memory formation of associative learning in Lymnaea.

The cellular basis of long-term memory (LTM) storage is not completely known. We have developed a preparation where we are able to specify that a single identified neuron, Right Pedal Dorsal 1 (RPeD1), is a site of LTM formation of associative learning in the pond snail, Lymnaea stagnalis. We demonstrated this by ablating the soma of the neuron but leaving behind its functional primary neurite, as evidenced by electrophysiological and behavioral analyses. The soma-less RPeD1 neurite continues to be a necessary participant in the mediation of aerial respiratory behavior, associative learning, and intermediate-term memory (ITM); however, LTM cannot be formed. However, if RPeD1's soma is ablated after LTM consolidation has occurred, LTM can still be accessed. Thus the soma of RPeD1 is a site of LTM formation.

Context extinction and associative learning in Lymnaea.

Aerial respiratory behavior in the pond snail Lymnaea was operantly conditioned so that snails learned not to perform aerial respiration in a hypoxic environment. Snails were trained in either the standard context (no food odorant) or a carrot (food-odorant) context. An operant training procedure of two 45-min training sessions with a 1-h interval between the sessions followed by a third 45-min training session 18 h later was sufficient to produce associative learning and long-term memory (LTM) that persisted for at least 5 days. If, however, following the third operant training session snails received three 45-min extinction training sessions, with each extinction session separated by at least a 1-h interval, LTM was not observed when tested the following day. That is, the memory was extinguished. Extinction, however, did not occur if the context of the extinction training was different from the context of the associative training. That is, in the snails trained in the standard context, extinction did not occur if the extinction training sessions were performed in the food-odorant context and vice versa.

The effects of continuous versus partial reinforcement schedules on associative learning, memory and extinction in Lymnaea stagnalis.

A continuous schedule of reinforcement (CR) in an operant conditioning procedure results in the acquisition of associative learning and the formation of long-term memory. A 50 % partial reinforcement (PR) schedule does not result in learning. The sequence of PR-CR training has different and significant effects on memory retention and resistance to extinction. A CR/PR schedule results in a longer-lasting memory than a PR/CR schedule. Moreover, the memory produced by the CR/PR schedule is resistant to extinction training. In contrast, extinction occurs following the PR/CR schedule.