Genes Upregulated by Operant Conditioning of Escape Behavior in the Pond Snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis is capable of learning by both classical conditioning and operant conditioning. Although operant conditioning related to escape behavior with punishment has been examined by some research groups, the molecular mechanisms are not known. In the present study, we examined changes in the expression levels of cAMP-response element binding protein 1 (CREB1), CREB2, CREB-binding protein (CBP), and monoamine oxidase (MAO) in the Lymnaea central nervous system (CNS) using real-time PCR following operant conditioning of escape behavior. CREB1 and CREB2 are transcription factors involved in long-term memory in Lymnaea; CBP is a coactivator with CREB1; and MAO is a degrading enzyme for monoamines (e.g., serotonin) with important roles in learning and memory in Lymnaea. In operant conditioning, the punishment cohort, in which snails escaping from the container encountered aversive KCl, exhibited significantly fewer escape attempts than the control cohort, in which snails escaping from the container encountered distilled water, during both the training and memory test periods. After the operant conditioning, CREB1 and CREB2 were upregulated, and the ratio of CREB1/CREB2 was also increased, suggesting that the operant conditioning of escape behavior involves these factors. MAO was also upregulated, suggesting that the content of monoamines such as serotonin in the CNS decreased. The upregulated genes identified in the present study will help to further elucidate learning and memory mechanisms in Lymnaea.

FOXO in Lymnaea: Its Probable Involvement in Memory Consolidation.

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.

Identification of Putative Molecules for Adiponectin and Adiponectin Receptor and Their Roles in Learning and Memory in Lymnaea stagnalis.

Adiponectin enhances insulin sensitivity, which improves cognition in mammals. How adiponectin affects the mechanism's underlying cognition, however, remains unknown. We hypothesized that experiments using the pond snail Lymnaea stagnalis, which has long been used in learning and memory studies and in which the function of insulin-like peptides affect learning and memory, could clarify the basic mechanisms by which adiponectin affects cognition. We first identified putative molecules of adiponectin and its receptor in Lymnaea. We then examined their distribution in the central nervous system and changes in their expression levels when hemolymph glucose concentrations were intentionally decreased by food deprivation. We also applied an operant conditioning protocol of escape behavior to Lymnaea and examined how the expression levels of adiponectin and its receptor changed after the conditioned behavior was established. The results demonstrate that adiponectin and adiponectin's receptor expression levels were increased in association with a reduced concentration of hemolymph glucose and that expression levels of both adiponectin and insulin-like peptide receptors were increased after the conditioning behavior was established. Thus, the involvement of the adiponectin-signaling cascade in learning and memory in Lymnaea was suggested to occur via changes in the glucose concentrations and the activation of insulin.

Changes in protein phosphorylation by insulin administration in the central nervous system of the gastropod mollusk Lymnaea stagnalis.

In the gastropod mollusk Lymnaea stagnalis, insulin-like peptides in the central nervous system (CNS) control behavioral changes associated with associative learning. Insulin administration to the Lymnaea CNS enhances the synaptic plasticity involved in this type of learning, but it has remained unclear which molecules in the insulin response cascade are involved. Here, to advance a comprehensive analysis, we used two-dimensional electrophoresis and comparative quantitative mass spectrometry to perform a protein analysis investigating the CNS molecules that respond to insulin administration. Our results revealed increased phosphorylation of AKT and RICTOR in the PI3K/AKT/mTOR signaling cascade and cytoskeleton-related proteins. Although it was expected that the molecules in the PI3K/AKT/mTOR signaling cascade were phosphorylated by insulin administration, our findings confirmed the correlation between insulin-induced phosphorylation of cytoskeleton-related proteins strongly involved in the synaptic changes and learning and memory mechanisms. These results contribute to elucidate the relationship between the insulin response and learning and memory mechanisms not only in Lymnaea but also in various invertebrates and vertebrates.

A choroid plexus cyst in the fourth ventricle of a Sprague-Dawley rat.

Choroid plexus cysts are rare lesions in the brain and are reported in humans and dogs. Herein, we report a choroid plexus cyst found in a 10-week-old female Sprague-Dawley rat. Histologically, a cyst measuring approximately 600 µm in diameter was found in the fourth ventricle of the brain. The cyst was lined with a single layer of flattened cells and was present in the connective tissue of the choroid plexus. Next to the cyst, a dilated tube was found with a similar morphology to the epithelium of the choroid plexus. Immunohistochemistry revealed that flattened cells lining the cyst were positive for cytokeratin and vimentin, and negative for GFAP and S-100, which is the same as in the normal choroid plexus, excluding vimentin. We diagnosed the present cyst as a spontaneously occurring choroid plexus cyst that was considered to be undergoing the epithelial-mesenchymal transition.