The transcription factor, CCAAT/enhancer-binding protein (C/EBP), is involved in important physiological processes, such as cellular proliferation and differentiation, homeostasis, and higher-order functions of the brain. In the present study, we investigated the distribution of mRNA and protein of C/EBP in the central nervous system of the pond snail, Lymnaea stagnalis, by in situ hybridization and immunohistochemistry. Specificity of the anti-mammalian C/EBP antibody against Lymnaea C/EBP (LymC/EBP) was confirmed by combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis or isoelectric focusing and immunoblotting. Cells positive for in situ hybridization were immunoreactive for LymC/EBP in all 11 ganglia. The motoneurons (B1, B2, B4, and B4 clusters) in the buccal ganglia and interneurons (cerebral giant cell, CGC) in the cerebral ganglia were positive for in situ hybridization and were immunopositive. In the pedal ganglion, the right pedal dorsal 1 (RPeD1), pedal A, and pedal C clusters exhibited positive signals of in situ hybridization and immunohistochemistry for LymC/EBP. CGC and RPeD1 are key neurons for associative learning. In addition, the neuropeptidergic cells in the cerebral, pleural, parietal, and visceral ganglia were positive for in situ hybridization and immunoreactive. Interestingly, although the cytoplasm of almost all immunopositive cells was stained, some neuropeptidergic cells located in the light parietal and visceral ganglia exhibited immunoreactivity in nuclei. Our results suggest that LymC/EBP is involved in learning and memory and in the expression and/or secretion of neuropeptides in Lymnaea.
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