Role of proteasome-dependent protein degradation in long-term operant memory in Aplysia.

We investigated the in vivo role of protein degradation during intermediate (ITM) and long-term memory (LTM) in Aplysia using an operant learning paradigm. The proteasome inhibitor MG-132 inhibited the induction and molecular consolidation of LTM with no effect on ITM. Remarkably, maintenance of steady-state protein levels through inhibition of protein synthesis using either anisomycin or rapamycin in conjunction with proteasome inhibition permitted the formation of robust 24 h LTM. Our studies suggest a primary role for proteasomal activity in facilitation of gene transcription for LTM and raise the possibility that synaptic mechanisms are sufficient to sustain 24 h memory.

Differential role of calpain-dependent protein cleavage in intermediate and long-term operant memory in Aplysia.

In addition to protein synthesis, protein degradation or protein cleavage may be necessary for intermediate (ITM) and long-term memory (LTM) to remove molecular constraints, facilitate persistent kinase activity and modulate synaptic plasticity. Calpains, a family of conserved calcium dependent cysteine proteases, modulate synaptic function through protein cleavage. We used the marine mollusk Aplysia californica to investigate the in vivo role of calpains during intermediate and long-term operant memory formation using the learning that food is inedible (LFI) paradigm. A single LFI training session, in which the animal associates a specific netted seaweed with the failure to swallow, generates short (30min), intermediate (4-6h) and long-term (24h) memory. Using the calpain inhibitors calpeptin and MDL-28170, we found that ITM requires calpain activity for induction and consolidation similar to the previously reported requirements for persistent protein kinase C activity in intermediate-term LFI memory. The induction of LTM also required calpain activity. In contrast to ITM, calpain activity was not necessary for the molecular consolidation of LTM. Surprisingly, six hours after LFI training we found that calpain activity was necessary for LTM, although this is a time at which neither persistent PKC activity nor protein synthesis is required for the maintenance of long-term LFI memory. These results demonstrate that calpains function in multiple roles in vivo during associative memory formation.

Protein phosphatase-dependent circadian regulation of intermediate-term associative memory.

The endogenous circadian clock is a principal factor modulating memory across species. Determining the processes through which the circadian clock modulates memory formation is a key issue in understanding and identifying mechanisms to improve memory. We used the marine mollusk Aplysia californica to investigate circadian modulation of intermediate-term memory (ITM) and the mechanisms through which the circadian clock phase specifically suppresses memory using the operant learning paradigm, learning that food is inedible. We found that ITM, a temporally and mechanistically distinct form of memory, is rhythmically expressed under light-dark and constant conditions when induced by either massed or spaced training. Strong circadian regulation of ITM occurs with memory exhibited only by animals trained during the early subjective day; no apparent memory is expressed when training occurs during the late subjective day or night. Given the necessity of multiple persistent kinase cascades for ITM, we investigated whether protein phosphatase activity affected circadian modulation. Inhibition of protein phosphatases 1 and 2A blocked ITM when animals were trained during the early (subjective) day while resulting in phase-specific memory rescue when animals were trained late in the subjective day and early night. In contrast, inhibition of calcineurin did not block ITM when animals were trained during the early day and permitted ITM when animals were trained during the late subjective day, early evening, and throughout the night. These results demonstrate that levels of protein phosphatase activity are critical regulators of ITM and one mechanism through which the circadian clock regulates memory formation.

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.