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.

SUMMARYThe 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.

SUMMARYAerial 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.

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 'highresponders 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.

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.