In Aplysia, serotonin mediates behavioral sensitization by increasing the strength of the synapse between sensory and motor neurons, a process known as facilitation. The retention of long-term facilitation is blocked by rapamycin, an inhibitor of a specific translational pathway. One possible rapamycinsensitive target is the increased translation of 5¢-terminal oligopyrimidine mRNAs. These transcripts encode components of the translational machinery and have been proposed to be important for retention of long-term facilitation. We have cloned the 5¢-terminal oligopyrimidine mRNA encoding eukaryotic elongation factor 2 and shown that serotonin increased its translation in synaptosomes. Another possible rapamycinsensitive target is the inactivation of eukaryotic elongation factor 2 kinase. Eukaryotic elongation factor 2 kinase phosphorylates and inactivates eukaryotic elongation factor 2, blocking translational elongation. Serotonin application decreased eukaryotic elongation factor 2 phosphorylation in synaptosomes and in isolated neurites, and this was blocked by rapamycin. We propose a role for the rapamycin-sensitive pathway in neurons. Stimulation blocks translation by inducing calcium entry and phosphorylation of eukaryotic elongation factor 2. This block is reversed through activation of the rapamycin-sensitive system and dephosphorylation of eukaryotic elongation factor 2. Keywords: Aplysia, eukaryotic elongation factor 2, rapamycin, S6 kinase, synaptic plasticity, translation. The marine mollusk, Aplysia californica, is a model system for the study of learning and memory. The memory for sensitization of the gill-and siphon-withdrawal reflexes in Aplysia is manifested on a cellular level by facilitation of synaptic strength at identified sensory-to-motor neuron synapses (Bailey and Chen 1983;Bailey et al. 1996). Experiments on cultured Aplysia sensory-motor neuron synapses have revealed the existence of a persistent phase of long-term facilitation (LTF) whose induction requires local translation. While LTF induced by somatic treatment with serotonin (5-hydroxytryptamine, 5-HT) lasts only 24 h, when 5-HT is also added to the synapse, facilitation persists for 72 h. A 5 min application of 5-HT to the synapse was sufficient to activate local translation and to allow for persistent LTF when gene expression was activated independently (Casadio et al. 1999). This persistent phase of LTF is blocked if the specific translational inhibitor, rapamycin, is applied locally to the synapse (Casadio et al. 1999). Addition of 5-HT to Aplysia sensory cell neurites or pleural ganglia has been shown to cause an increase in the rate of translation that is sensitive to rapamycin (Yanow et al. 1998;Casadio et al. 1999), suggesting that a local rapamycin-sensitive up-regulation of protein synthesis may be specifically required for the retention of plasticity. Recently, a rapamycin-sensitive increase in the levels of the cytoplasmic polyadenylation element binding protein (CPEB) was shown to be an important component of t...

The target of rapamycin (TOR) plays an important role in memory formation in Aplysia californica. Here, we characterize one of the downstream targets of TOR, the eukaryotic initiation factor 4E (eIF4E) binding protein (4EBP) from Aplysia. Aplysia 4EBP contains the four critical phosphorylation sites regulated by TOR as well as an N-terminal RAIP motif and a C-terminal TOS site. Aplysia 4EBP was hypophosphorylated in synaptosomes, and serotonin addition caused a rapamycin-sensitive increase in 4EBP phosphorylation both in synaptosomes and in isolated neurites. Aplysia 4EBP was regulated in a fashion similar to that of mammalian 4EBPs, binding to eIF4E when dephosphorylated and releasing eIF4E after phosphorylation. Overexpression of 4EBP in the soma of Aplysia neurons caused a specific decrease in cap-dependent translation that was rescued by concomitant overexpression of eIF4E. However, eIF4E overexpression by itself did not increase cap-dependent translation, suggesting that increasing levels of free eIF4E by phosphorylating 4EBP is not important in regulating cap-dependent translation in the cell soma. Total levels of eIF4E were also regulated by 4EBP, suggesting that 4EBP can also act as an eIF4E chaperone. These studies demonstrate the conserved nature of 4EBP regulation and its role in cap-dependent translation and suggest differential roles of 4EBP phosphorylation in the soma and synapse.An increase in the rate of translation that is sensitive to the pharmacological agent rapamycin was recently shown to be an important event underlying lasting phases of synaptic plasticity (2,5,16,58). The primary target of rapamycin in cells is the target of rapamycin (TOR) (9,24,30). One way in which TOR controls translation is by regulating the phosphorylation state of the eukaryotic initiation factor 4E (eIF4E) binding protein (4EBP). Dephosphorylated 4EBP tightly binds eIF4E and represses cap-dependent protein synthesis, and TOR-dependent phosphorylation removes this repression (6,11,46). The amount of eIF4E available in the cell can be a rate-limiting factor in cell growth since its overexpression causes an increase in the expression of growth-promoting proteins, such as ornithine decarboxylase, cyclin D1, and c-Myc (15,49,51,55). Therefore, activation of TOR may contribute to enduring forms of synaptic plasticity through phosphorylation of 4EBP and an increase in translation of molecules that contribute to synaptic growth and/or reorganization.4EBP is located at synaptic sites in mammalian dendrites (58) and is phosphorylated in a TOR-dependent manner after brain-derived neurotrophic factor treatment in dendrites or late-phase long-term potentiation (LTP)-inducing stimuli (3, 35, 57). Moreover, knocking out the gene for 4EBP2, the major 4EBP isoform in adult neurons, converts early LTP into late LTP in the Schaffer collateral pathway (3), suggesting that activating translation through 4EBP phosphorylation is a critical step in generating long-term changes. Long-term depression induced by the addition of an mGLUR ago...