Persistently Increased Expression of PKMzeta and Unbiased Gene Expression Profiles Identify Hippocampal Molecular Traces of a Long‐Term Active Place Avoidance Memory and “Shadow” Proteins

Long-term memory formation transiently activates Ca²⁺-calmodulin kinase II and atypical protein kinase C isoform iota/lambda, whereas persistent activation of the other atypical PKC, protein kinase M zeta (PKMζ), together with its interacting partner, the scaffolding-protein KIBRA (Wwc1), are necessary for maintaining potentiated synapses and memory. Here, we use immediate-early gene (IEG) Arc activation during active place avoidance memory expression to tag memory-activated neurons with EYFP-ChR2. PKMζ immunohistochemistry identified persistently altered hippocampal somatodendritic domains. EYFP-PKMζ colocalization persistently increases in the hippocampal trisynaptic pathway (dentate gyrus [DG]→CA3→CA1) tracing a 1-month PKMζ engram. DG, CA3, and CA1 transcriptional profiling identifies that memory persistence correlates with upregulated IEGs Arc, Fos, and NPas4 in DG, but not with Prkcz, the PKMζ gene, or most genes known to be crucial for LTP and memory. This rules out strong memory-related transcriptional but not translational regulation or altered stability of such "shadow proteins" like PKMζ that, despite being crucial for memory maintenance, evade detection by unbiased transcriptome profiling. In contrast, our method Correlation Signal Co-cluster Reduction (C-SCoRe) incorporates weak linear and non-linear gene correlations and highlights network interaction changes predicting memory, and related IEG and Prkcz/Wwc1 expression. Manifold transcriptional relationships can reveal shadow molecular components of long-term memory.