Learning-specific remodeling of the hippocampal palmitoylome

Protein S-palmitoylation is a reversible lipid modification that regulates protein trafficking, membrane association, and synaptic signaling, yet its role in learning-induced neuronal plasticity remains incompletely understood. Here, we investigated how spatial learning remodels the hippocampal palmitoylome in rats trained in the Morris water maze using short-term (STT; one session, 15 trials; probe at 1 h) or long-term (LTT; four sessions over four days; probe at 24 h) paradigms. Palmitoylated proteins were profiled by acyl-biotin exchange coupled with tandem mass tag labeling and LC-MS/MS. We identified 5,260 proteins, including 763 palmitoylated species. Spatial learning induced extensive remodeling of protein S-palmitoylation, with markedly greater changes after STT than LTT. Using yoked controls, we distinguished palmitoylation changes associated with learning the hidden platform location from those induced by general behavioral experience. Comparison of trained and yoked animals identified 186 and 62 differentially palmitoylated proteins after STT and LTT, respectively, whereas yoked animals also exhibited extensive changes relative to naive controls, demonstrating that behavioral experience alone substantially reshapes the hippocampal palmitoylome. Functional enrichment revealed that STT preferentially engaged pathways related to synaptic transmission, cytoskeletal remodeling, GTPase signaling, and cellular metabolism, whereas LTT was associated with protein translation and synaptic organization. Site-specific analysis identified numerous previously unreported palmitoylation sites. Hierarchical analysis further identified diacylglycerol lipase- (DAGLA) as the only protein whose palmitoylation consistently reflected both general Morris water maze experience and learning the hidden platform location across both paradigms. Together, these findings establish S-palmitoylation as a dynamic regulator of experience-dependent hippocampal plasticity.