Massed training-induced intermediate-term operant memory in aplysia requires protein synthesis and multiple persistent kinase cascades.

The Aplysia feeding system with its high degree of plasticity and well characterized neuronal circuitry is well suited for investigations of memory formation. We used an operant paradigm, learning that food is inedible (LFI), to investigate the signaling pathways underlying intermediate-term memory (ITM) in Aplysia. During a single massed training session, the animal associates a specific seaweed with the failure to swallow, generating short-term (30 min) and long-term (24 h) memory. We investigated whether the same training protocol induced the formation of ITM. We found that massed LFI training resulted in temporally distinct protein synthesis-dependent memory evident 4-6 h after training. Through in vivo experiments, we determined that the formation of ITM required protein kinase A, protein kinase C, and MAPK. Moreover, the maintenance of ITM required PKA, PKM Apl III, and MAPK because inhibition of any of these kinases after training or before testing blocked the expression of memory. In contrast, additional experiments determined that the maintenance of long-term memory appeared independent of PKM Apl III. Using Western blotting, we found that sustained MAPK phosphorylation was dependent upon protein synthesis, but not PKA or PKC activity. Thus, massed training-induced intermediate-term operant memory requires protein synthesis as well as persistent or sustained kinase signaling for PKA, PKC, and MAPK. While short-, intermediate-, and long-term memory are induced by the same training protocol, considerable differences exist in both the combination and timing of signaling cascades that induce the formation and maintenance of these temporally distinct memories.