FOXO in Lymnaea: Its Probable Involvement in Memory Consolidation

SIMPLE SUMMARY: Insulin in the central nervous system (CNS) affects learning and memory, but the involvement of forkhead box O (FOXO), a transcription factor belonging to the insulin signaling cascade, is unclear. We identified FOXO in the pond snail Lymnaea stagnalis and examined its subcellular localization in CNS neurons. In the CNS, FOXO was observed in neuronal nuclei in food-deprived snails and in the neuronal cytoplasm of food-satiated snails. FOXO translocated to the nuclei in food-satiated snails when they were subjected to conditioned taste aversion (CTA) learning. Although insulin administration to CNSs isolated from food-satiated snails was expected to induce the translocation of FOXO into nuclei, FOXO remained in the cytoplasm. In addition, insulin administered to the CNSs upregulated only the expression of FOXO mRNA and not its related molecules. Together, these findings suggest that food deprivation prepares FOXO to function in neuronal nuclei and enhances CTA learning in snails. Insulin administration, however, may stimulate other pathways that are not downstream of the FOXO response cascade. ABSTRACT: Food deprivation activates forkhead box O (FOXO), a transcription factor downstream of insulin receptors. In the pond snail Lymnaea stagnalis, insulin signaling and food deprivation improve memory consolidation following conditioned taste aversion (CTA) learning. We investigated the subcellular localization of FOXO in Lymnaea and changes in its expression levels following food deprivation, CTA learning, and insulin administration. Immunohistochemistry revealed that Lymnaea FOXO (LymFOXO) was located in the central nervous system (CNS) neuronal cytoplasm in food-satiated snails but was mainly in neuronal nuclei in food-deprived snails. Following CTA acquisition, LymFOXO translocated to the nuclei in food-satiated snails and remained in the nuclei in food-deprived snails. Contrary to our expectations, insulin administered to the CNS did not induce LymFOXO translocation into the nuclei in food-satiated snails. Real-time PCR was used to quantify LymFOXO mRNA levels, its target genes, and insulin signaling pathway genes and revealed that LymFOXO mRNA was upregulated in food-deprived snails compared to food-satiated snails. Insulin applied to isolated CNSs from food-satiated snails increased LymFOXO compared to vehicle-treated samples. Food deprivation prepares FOXO to function in the nucleus and enhances CTA learning in snails. Insulin application did not directly affect LymFOXO protein localization. Thus, insulin administration may stimulate pathways other than the LymFOXO cascade.