Associative memory in three aplysiids: correlation with heterosynaptic modulation

Abstract

Much recent research on mechanisms of learning and memory focuses on the role of heterosynaptic neuromodulatory signaling. Such neuromodulation appears to stabilize Hebbian synaptic changes underlying associative learning, thereby extending memory. Previous comparisons of three related sea-hares (Mollusca, Opisthobranchia) uncovered interspecific variation in neuromodulatory signaling: strong in Aplysia californica, immeasureable in Dolabrifera dolabrifera, and intermediate in Phyllaplysia taylori. The present study addressed whether this interspecific variation in neuromodulation is correlated with memory of associative (classical conditioning) learning. We differentially conditioned the tail-mantle withdrawal reflex of each of the three species: Mild touch to one side of the tail was paired with a noxious electrical stimulus to the neck. Mild touch to the other side served as an internal control. Post-training reflex amplitudes were tested 15-30 min after training and compared with pre-test amplitudes. All three species showed conditioning: training increased the paired reflex more than the unpaired reflex. However, the temporal pattern of conditioning varied between species. Aplysia showed modest conditioning that grew across the post-test period. Dolabrifera showed distinctly short-lived conditioning, present only on the first post-test. The time course of memory in Phyllaplysia was intermediate, although not statistically distinguishable from the other two species. Taken together, these experiments suggest that evolutionary changes in nonassociative heterosynaptic modulation may contribute to evolutionary changes in the stability of the memory of classical conditioning.

Figure 1.

Aplysia showed differential conditioning that was not expressed until 30 min after training. Shown are mean (± SEM) tail-mantle withdrawal durations before (negative time) and after (positive time) differential conditioning consisting of five pairings (20 min) of a tactile tail stimulus (paired) with noxious electric shock to the neck. Unpaired tactile stimuli were delivered exactly between the paired stimuli. The dashed line with triangles indicates the calculated difference between the reflex withdrawal of the paired and unpaired stimuli. + Indicates marginal statistical difference from pre-training baseline (repeated measures t-test with Bonferroni correction, two-tailed, P ≤ 0.05).

Figure 2.

Dolabrifera showed significant differential conditioning that lasted no more than 20 min after training. Only the first post-test showed a significant difference between the paired and unpaired reflexes. * Indicates statistical difference from pre-training baseline (repeated measures t-test with Bonferroni correction, two-tailed, P ≤ 0.05).

Figure 3.

Phyllaplysia showed significant differential conditioning immediately after training that disappeared by 25–30 min after training. Only the first post-test showed a significant difference between the paired and unpaired reflexes. Asterisks indicate statistical difference from pre-training baseline (two-tailed probabilities: *P ≤ 0.05; **P ≤ 0.01) using a repeated measures t-test with Bonferroni correction.

Figure 4.

Memory for classical conditioning was heterogenous across species: Aplysia (diamonds), Dolabrifera (squares), Phyllaplysia (triangles). Mean reflex differential (Paired minus Unpaired, standardized by mean maximum for each species). Data are derived from the difference scores in Figures 1, 2, 3 to facilitate comparison. See those figures to determine variation around each score. Dolabrifera and Phyllaplysia showed significant learning, but only 15 min after training. Aplysia also showed significant learning averaged over all four post-tests. Symbols indicate statistical difference from pre-training baseline (two-tailed probabilities: +P ≤ 0.10; **P ≤ 0.01) using a repeated measures t-test with Bonferroni correction.