Expression, phosphorylation, and glycosylation of CNS proteins in aversive operant conditioning associated memory in Lymnaea stagnalis.

Long-term memory formation requires "de novo" expression and post-translational modification of many proteins. Understanding the temporal and spatial regulatory pattern of these proteins is fundamental to decoding the molecular basis of learning and memory. We characterized changes in expression, phosphorylation, and glycosylation of CNS proteins after operant conditioning in pond snail Lymnaea stagnalis. The phosphorylation and the glycosylation levels of proteins, measured by the ratio of Pro-Q Diamond (phosphoproteins) or Pro-Q Emerald (glycoproteins) vs. SYPRO-Ruby (total proteins) signals, increased during memory formation. Proteins whose modulation of phosphorylation might be involved in learning and memory were identified by mass spectrometry (MS) and are associated with cytoskeleton, glutamine cycle, energy metabolism, G-protein signaling, neurotransmitter release regulation, iron transport, protein synthesis, and cell division. Phosphorylation of actin increased during memory formation. To identify proteins whose expression levels changed in long-term memory formation we used two-dimensional difference gel electrophoresis followed by MS. The up-regulated proteins are mostly associated with lipoprotein and cholesterol metabolism, protein synthesis and degradation, cytoskeleton, nucleic acid synthesis, and energy supply. The down-regulated proteins are enzymes of aspartic acid metabolism involved in regulation of protein synthesis. Our proteomic analyses have revealed a number of candidate proteins associated with memory formation. These findings provide new directions for further investigation into the signaling networks required for memory formation and consolidation.