Habituation-Induced Expression of Immediate Early Genes in the Octopus Brain

Understanding the neural basis of learning and memory in cephalopods remains challenging because of their behavioral complexity and the limited reproducibility of memory tasks. Here, I present a combined behavioral and molecular analysis using a simple habituation paradigm, Training Response to Artificial Prey (TRAP), in Octopus bimaculoides. Subjects showed a rapid, stimulus-specific decrease in exploration of a neutral prey-like object across trials. Habituation occurred in 100% of two- and six-month-old subjects, and multiple daily trials induced long-term memory lasting up to five days. Using whole-mount Hybridization Chain Reaction (HCR), I detected spatial activation of candidate immediate early genes, creb and egr1, during early memory consolidation. 15 minutes after learning, both genes were upregulated across the central brain, mainly in the subesophageal lobes, and axial ganglia. At 30 minutes, expression spread to the supraesophageal region, notably the vertical lobe, consistent with regions implicated in long-term memory. These results demonstrate a sequential activation from arms to subesophageal and supraesophageal regions, supporting proposed models of distributed information processing in the octopus nervous system. HIGHLIGHTSO_LITraining Response to Artificial Prey (TRAP) is a simple and highly repeatable method to evaluate memory in octopuses. C_LIO_LIRepeated trials induced robust, long-term habituation lasting up to five days. C_LIO_LIImmediate early genes creb and egr1 were rapidly upregulated after training. C_LIO_LI(d) Gene expression spread sequentially across nervous system regions recruited for memory formation. C_LI