A homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide is both necessary and instructive for the rapid formation of associative memory in an invertebrate

Abstract

Similar to other invertebrate and vertebrate animals, cAMP-dependent signaling cascades are key components of long-term memory (LTM) formation in the snail Lymnaea stagnalis, an established experimental model for studying evolutionarily conserved molecular mechanisms of long-term associative memory. Although a great deal is already known about the signaling cascades activated by cAMP, the molecules involved in the learning-induced activation of adenylate cyclase (AC) in Lymnaea remained unknown. Using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy in combination with biochemical and immunohistochemical methods, recently we have obtained evidence for the existence of a Lymnaea homolog of the vertebrate pituitary adenylate cyclase-activating polypeptide (PACAP) and for the AC-activating effect of PACAP in the Lymnaea nervous system. Here we first tested the hypothesis that PACAP plays an important role in the formation of robust LTM after single-trial classical food-reward conditioning. Application of the PACAP receptor antagonist PACAP6-38 around the time of single-trial training with amyl acetate and sucrose blocked associative LTM, suggesting that in this "strong" food-reward conditioning paradigm the activation of AC by PACAP was necessary for LTM to form. We found that in a "weak" multitrial food-reward conditioning paradigm, lip touch paired with sucrose, memory formation was also dependent on PACAP. Significantly, systemic application of PACAP at the beginning of multitrial tactile conditioning accelerated the formation of transcription-dependent memory. Our findings provide the first evidence to show that in the same nervous system PACAP is both necessary and instructive for fast and robust memory formation after reward classical conditioning.

Figure 1.

PACAP6-38 blocks the formation of memory after both single-trial chemical (a “strong” training paradigm) and multitrial tactile classical conditioning (a “weak” training paradigm) of feeding. A, Application of this PACAP receptor antagonist is effective in blocking memory formation when applied 120 min before or 8 min after single-trial chemical classical conditioning of feeding, but is ineffective when applied at 6 h after training. The means of the CS-evoked feeding responses and the SEM are shown in this diagram and all subsequent figures. In this figure, the mean baseline feeding response is shown as a dashed line and the SEM of the baseline response is shown as a gray band. Asterisks indicate significant differences from the unpaired baseline level. Test statistics were as follows: one-way ANOVAs: injection 120 min before training, F_(2,70) = 23.8, p < 0.0001; Tukey's test between paired + saline and unpaired baseline, p < 0.001; Tukey's test between paired + PACAP6-38 and unpaired baseline, p > 0.05 (n.s.). Injection 8 min after training, F_(2,49) = 13.9, p < 0.0001, Tukey's test between paired + saline and unpaired baseline, p < 0.001; Tukey's test between paired + PACAP6-38 and unpaired baseline, p > 0.05 (n.s.). Injection 6 h after training, F_(2,52) = 16.3; Tukey's test between paired + saline and unpaired baseline, p < 0.001; Tukey's test between paired + PACAP6-38 and unpaired baseline, p < 0.001. B, PACAP6-38 is also effective in blocking memory formation after multitrial tactile classical conditioning of feeding. The responses are calculated as differences between the spontaneous rasping rates in water and the rasping rates after the delivery of the conditioned stimulus. Without training, lip touch has a mild inhibitory effect on spontaneous rasping (Kemenes and Benjamin, 1989), so in the case of tactile stimulation, the rates after the application of the stimulus can also take negative values (also see Figs. 2–5). Test statistics were as follows: one-way ANOVA, F_(2,57) = 8.9, p < 0.0004; Tukey's test between paired + saline and unpaired baseline, p < 0.01; Tukey's test between paired + PACAP6-38 and unpaired baseline, p > 0.05 (n.s.). Asterisks indicate significant differences from the unpaired baseline level.

Figure 2.

Exogenous PACAP38 boosts associative memory expressed early (90 min) after multitrial tactile classical conditioning (a “weak” training paradigm). A, Timeline of the injection (PACAP or saline), training trials (lip touch + sucrose) and test (lip touch only) protocol. B, Feeding responses to the test lip touch in the paired, unpaired and naive group of animals. Test statistics were as follows: two-way ANOVA for interaction, F_(2,66) = 4.6, p < 0.01; two-way ANOVA for training and injection, all differences are n.s. (F_(2,66) = 1.3 and 1.8, p = 0.27 and 0.18, respectively). The lack of a significant effect of the training factor means that without PACAP treatment, there is no significant memory expression at 90 min even when the animals have been classically conditioned. The lack of a significant effect of the injection factor, on the other hand, means that PACAP treatment alone does not result in an enhanced feeding response to the CS. Bonferroni's post hoc tests confirmed that, when compared against the effect of saline, PACAP only had a significant effect in the paired group (t = 3.6, p < 0.01). Unpaired group, t = 0.7, p > 0.05 (n.s.); naive group, t = 0.0, p > 0.05 (n.s.). The asterisk indicates the significant change resulting from the interaction between PACAP treatment and classical conditioning.

Figure 3.

Exogenous PACAP38 boosts memory expressed late (18 h) after multitrial tactile classical conditioning. The injection and training protocol on each of the 3 d of the experiment was the same as that shown in Figure 2A, but the test (lip touch only) was performed 18 h after the last trial. The graphs show the feeding responses to the test lip touch in paired and unpaired groups of animals after increasing numbers of trials or at corresponding time points in the naive control group (indicated by the number of trials shown between inverted commas). Test statistics were as follows: two-way ANOVA for interaction, 3 trials + 18 h, F_(2,66) = 5.6, p < 0.005, 6 trials + 18 h, F_(2,66) = 4.7, p < 0.01, 9 trials + 18 h, F_(2,64) = 3.9, p < 0.02; two-way ANOVA for training, 3 trials + 18 h and 6 trials + 18 h, F_(2,66) = 1.3 and 1.9, p = 0.27 and 0.14, respectively (n.s.), 9 trials + 18 h, F_(2,64) = 13.6, p < 0.0001; two-way ANOVA for injection, all differences are n.s. (3 trials, F_(2,66) = 1.8, p = 0.18; 6 trials, F_(2,66) = 1.5, p = 0.22; 9 trials, F_(2,64) = 0.7, p = 0.39). The lack of a significant effect of the training factor in the experiments with 3 and 6 trials means that without PACAP treatment, there is no significant memory expression at 18 h even when the animals have been classically conditioned. The lack of a significant effect of the injection factor in all three experiments (3, 6, and 9 trials) means that PACAP treatment alone does not result in an enhanced feeding response to the CS. Bonferroni's post hoc tests confirmed that, when compared against the effect of saline, PACAP only had a significant effect in the paired groups (3 trials, t = 3.8, p < 0.01; 6 trials, t = 3.5, p < 0.01; 9 trials, t = 2.9, p < 0.05). Unpaired groups, 3 trials, t = 0.8, p > 0.05 (n.s.); 6 trials, t = 0.8, p > 0.05 (n.s.); 9 trials, t = 1.09, p > 0.05 (n.s.). Naive groups, “3 trials”, t = 0.2, p > 0.05 (n.s.); “6 trials”, t = 0.1, p > 0.05 (n.s.); “9 trials”, t = 0.0, p > 0.05 (n.s.). Asterisks indicate significant changes resulting from the interaction between PACAP treatment and classical conditioning. The hash symbol indicates the significant effect arising solely from classical conditioning after 9 trials. One-way ANOVA, F_(2,34) = 6.7, p < 0.04. Tukey's post hoc tests, paired versus unpaired, p < 0.05; paired versus naive, p < 0.01; unpaired versus naive, p > 0.05 (n.s.).

Figure 4.

Both the early and late memory-boosting effects of exogenously applied PACAP38 are blocked by actinomycin-D. Memory tests were performed at 90 min and 18 h after 3 trials. Test statistics were as follows: 90 min test, one-way ANOVA F_(2,50) = 17.5, p < 0.0001; Tukey's (PACAP vs both PACAP + Act-D and saline), p < 0.001. 18 h test, one-way ANOVA F_(2,50) = 4.1, p < 0.02; Tukey's (PACAP + saline versus both PACAP + Act-D and saline), p < 0.05.

Figure 5.

The memory-boosting effects of exogenously applied PACAP38 are blocked by the PACAP receptor antagonist PACAP6-38. Memory tests were performed at 90 min and 18 h after 3 trials. Test statistics were as follows: 90 min test, one-way ANOVA F_(2,38) = 6.5, p < 0.004, Tukey's (PACAP + saline versus both PACAP + antagonist and saline + saline), p < 0.05. 18 h test, one-way ANOVA F_(2,38) = 7.6, p < 0.002, Tukey's (PACAP + saline versus both PACAP + antagonist and saline + saline), p < 0.05.

Figure 6.

A schematic representation of the signaling molecules, receptors, ion channels, and pathways involved in memory formation after food-reward classical conditioning in Lymnaea. Molecular components examined in the present study (PACAP, PAC1) as well as those identified in previous reports [AC, Pirger et al. (2010); C/EBP, Hatakeyama et al. (2004); CaMKII and NMDA, Wan et al. (2010); cAMP, Nikitin et al. (2006); CREB, Ribeiro et al. (2003) and Sadamoto et al. (2004); MAPK, Ribeiro et al. (2005); Na⁺, Ca²⁺, and K⁺ channels, Staras et al. (2002) and Nikitin et al. (2008); NOS and NO, Kemenes et al. (2002) and Korneev et al. (2005); PKA, G. Kemenes et al. (2006); RNA and protein synthesis, Fulton et al. (2005); sGC, Ribeiro et al. (2008) and Michel et al. (2008)], are shown boxed. G, G-Protein; DA, dopamine; 5-HT, serotonin; MAPK, mitogen-activated protein kinase; CaMKII, calcium/calmodulin-dependent kinase II; NOS, nitric oxide synthase; NO, nitric oxide; sGC, soluble guanylyl cyclase; PKG, cGMP-activated protein kinase; PCREB, phosphorylated cAMP response element-binding protein; C/EBP, CCAAT enhancer-binding protein.