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.

Juvenile Lymnaea ventilate, learn and remember differently than do adult Lymnaea.

Adult snails are capable of learning associatively not to perform aerial respiration and then to consolidate the acquired behaviour into long-term memory (LTM). Juvenile Lymnaea, however, perform aerial respiration significantly less often and the three-neuron circuit that drives this behaviour operates significantly differently than in it does in adults. We asked whether these ontogenic behavioural and neurophysiological differences are manifested as an altered ability of juveniles to learn and/or form LTM. We found that juvenile snails learn significantly less well than adults and are, as a group, incapable of forming LTM. To control for the possibility that the poor learning and inability to form memory were the result of juvenile's receiving on average fewer reinforcing stimuli because they perform aerial respiration less often than adults we subjected juveniles to an enforced period of hypoxia to "motivate" juveniles. Motivated juveniles perform aerial respiration as often as adults; yet these "motivated" juveniles continue to be poor learners and still cannot form LTM. Additionally, a small percentage of juveniles perform aerial respiration as often as adults (i.e. high responders). When these "high-responders" were trained they still exhibited poorer learning ability compared with adults and could not form LTM. We conclude that juvenile snails have a more difficult time learning and remembering to suppress aerial respiratory activity than do adults.

Operant conditioning of an in vitro CNS-pneumostome preparation of Lymnaea.

Operant conditioning of aerial respiratory behaviour and its consolidation into long-term memory in Lymnaea has been previously studied in both intact, freely moving snails and in in vitro preparations made from previously trained snails. Here, we show in previously untrained semi-intact in vitro Lymnaea preparations that aerial respiratory behaviour can also be operantly conditioned. Neither yoked control nor 'run-down' control procedures in these in vitro preparations result in an alteration of aerial respiratory behaviour. Memory in the operantly trained semi-intact preparations persists for at least 1h after training. Intracellular recordings made from RPeD1, one of the 3-CPG neurons and the neuron that initiates CPG activity; show that there are specific changes in central excitatory input to this neuron concurrent with learning and its consolidation into memory. In addition following the acquisition of learning and its consolidation into memory the ability of RPeD1 and VI/J neurons when depolarized to cause a pneumostome opening is significantly decreased. Thus, previously untrained in vitro semi-intact preparations can be used to study changes in neuronal activity in a neuron known to be both necessary for the behaviour and for memory formation.

Forgetting and the extension of memory in Lymnaea.

Aerial respiratory behaviour in Lymnaea stagnalis was operantly conditioned using a procedure that results in long-term memory (LTM) persisting for 1 but not 3 days. By manipulating the snails' post-training environment, i.e. preventing Lymnaea from performing aerial respiratory behaviour, memory persistence was significantly extended. Memory retention, however, is only extended if snails are prevented from performing aerial respiration in the same context in which they were trained. Snails trained in the 'standard' context but prevented from performing aerial respiration in the 'carrot-odor' context (and vice versa) did not extend their memory. These data are consistent with the hypothesis that forgetting is due to interfering events, that occur following learning and memory consolidation.

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.

Electrophysiological differences in the CpG aerial respiratory behavior between juvenile and adult Lymnaea.

Intact, freely moving juvenile Lymnaea perform aerial respiration significantly less often than do adults. We therefore hypothesized that RPeD1, the central pattern generator (CPG) neuron that initiates rhythmogenesis, would be less active in juveniles than adults. Using both isolated and semi-intact preparations to directly test this hypothesis, we found the opposite; juvenile RPeD1s were significantly smaller and more excitable than RPeD1s from adults. Significant age-related differences were found in the membrane resistance (greater in juveniles), time constant (smaller in juveniles), and rheobase current (lower in juveniles), all of which would tend to make juvenile cells significantly more excitable. However, there were significant age-related differences in the synaptic connectivity within the CPG and in peripheral input to the CPG, all which favor more rhythmic activity in the adult CPG. As was the case for intact Lymnaea, juvenile semi-intact preparations perform aerial respiration less often than do adults. The difference in excitability between juvenile and adult RPeD1s is therefore not sufficient to cause increased rhythmogenesis. Age-related changes in synaptic connectivity within the respiratory CPG and in peripheral modulation allow respiratory rhythmogenesis to be more easily expressed in adults which may compensate for their decreased dependence on cutaneous respiration as their surface to volume ratio changes as the grow in size.

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.

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.