Thirty-Two Days of Fear: Long-Term Memory Retention and Neural Activation in Amphibians

Long-term memory retention is essential for adaptive behavior, yet its persistence and neural correlates remain poorly understood in amphibians. We investigated the temporal stability of aversive memories and associated neural activation in Rhinella arenarum. Toads underwent six days of classical conditioning, in which a 300 mM NaCl solution (CS) was paired with an aversive 800 mM NaCl solution (US). Heart rate was recorded through implanted dermal electrodes as a physiological measure of the conditioned response. Independent groups were tested for memory retention 1, 4, 8, 16, 32, or 64 days after acquisition. Conditioned animals showed a significant anticipatory cardiac response to the CS compared with controls at 1, 4, 8, 16, and 32 days, whereas this difference was no longer observed at 64 days. Neural activity assessed through AgNOR staining showed a similar temporal pattern, with increased activation in the amygdala associated with aversive learning up to 32 days, but not at the 64-day retention test. These findings reveal long-lasting but temporally limited aversive memory in amphibians and provide evidence of neural changes associated to its persistence.