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

Ken Lukowiak^*, Susan Sangha, Chloe McComb, Nishi Varshney, David Rosenegger, Hisayo Sadamoto and Andi Scheibenstock

Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada, T2N 4N1

View larger version (54K): [in a new window]   Fig. 1. Lymnaea, the pneumostome and training. (A) Cartoon of the training apparatus used to operantly condition aerial respiratory behaviour in Lymnaea. A beaker is filled with pond-water and then N2 is bubbled through it for 20 min in order to make the pond water hypoxic. Snails are then added, given a 10-min acclimatization period and then training begins. N2 is continuously bubbled throughout the training period. Each time the snail attempts to open its pneumostome, the respiratory orifice, it receives a tactile stimulus to the pneumostome area, which causes the pneumostome to close. The tactile stimulus is delivered by means of a sharpened wooden stick, the `digital stimulator'. (B) Photograph of a 2.5 cm (i.e. adult) snail. The pneumostome (arrow) is open. The pneumostome only opens when the snail is at the air–water interface.

View larger version (26K): [in a new window]   Fig. 2. Soma ablation in Lymnaea. (A) Photomicrograph of a Lucifer yellow-filled RPeD1. RPeD1 sends neuritic branches to other ganglia, where it synapses with other members of the aerial respiratory network, and also sends processes to the pneumostome area, where it receives both tactile and chemosensory input. The soma diameter of RPeD1 is approximately 75 µmol l-1. (B) A similar RPeD1 Lucifer yellow fill, except that gentle poking of it with a glass microelectrode has ablated its soma. The isolated neurite remains viable, as detected electrophysiologically for at least 2 weeks and behaviourally for at least 1 month. (C) A cartoon of the aerial respiratory central pattern generator (CPG; RPeD1, VD4 and IP3; see Syed et al., 1990) showing how synaptic connections are made in these unipolar neurons. Removal of RPeD1's soma does not disrupt either the pre- or postsynaptic specialization areas of the neuron. The closed circles represent an inhibitory chemical synaptic input, the bars represent excitatory synaptic input, and a combined bar and filled circle represents a conjoint inhibitory/excitatory input. All synapses are chemical and involve, at a minimum, the classical transmitters acetylcholine and dopamine and peptide transmitters of the RFamide family.

View larger version (12K): [in a new window]   Fig. 3. RPeD1 soma ablation and memory formation. Ablating RPeD1 somata 2 days before training does not affect the snails' (N=20) ability to learn. That is, there is a significant difference between Session 1 and Session 2. In addition, these snails (N=20) can form intermediate term memory (ITM). That is, in a memory test (MT) 2 h after the last training session, the criteria for memory are met. However, if the memory is tested 24 h later in these same snails (N=20), the criteria for memory are not met. We conclude that RPeD1 soma-ablated snails do not form long-term memory (LTM).

View larger version (12K): [in a new window]   Fig. 4. RPeD1 soma-ablated snails can still access long-term memory (LTM). A cohort (N=10) of snails received two 45-min training sessions, with a 1 h interval between the sessions. One hour following Session 2, all snails had their RPeD1 soma ablated (RPeD1 SOAB). When we tested for memory 3 days later (MT), it was present. That is, the criteria for memory were met. However, to be certain that the apparent memory was truly memory and not unresponsiveness of the snails, they were challenged 1 h later with a 45-min change-of-context test (CC). These snails are still capable of responding, and thus RPeD1 soma-ablated snails can still access previously encoded LTM.

View larger version (12K): [in a new window]   Fig. 5. Learning, memory and the assignment of marks. (A) The learning and memory curves for snails (N=1490) that receive two 45-min training sessions, with an interval of 1 h between the two training sessions. Memory (MT) was tested 24 h later in 490 of these snails. The other 1000 snails were used in extinction, forgetting, change-of-context and memory-extension experiments and thus could not contribute to the 24 h memory test. An analysis of variance (ANOVA; F1490,1=1460, P<0.0001) shows that learning occurred. When we tested for memory 24 h later we found that the number of attempted pneumostome openings in the memory test session was not significantly greater (P>0.05) than the number in Session 2 and was significantly less than the number in Session 1 (P<0.001). Thus, the criteria for memory were met. (B) The assignment of marks to individual snails. The vast majority of snails (77%) received either an A or a B mark, while 12% `flunked' (i.e. received an F).